PGC-1, a novel brown fat PPARγ coactivator

ABSTRACT

The invention provides isolated nucleic acids molecules, designated PGC-1 nucleic acid molecules, which encode proteins which can modulate various adipocyte-associated activities including, for example, thermogenesis in adipocytes, e.g., brown adipocytes, and adipogenesis. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing PGC-1 nucleic acid molecules, host cells into which the expression vectors have been introduced, and nonhuman transgenic animals in which a PGC-1 gene has been introduced or disrupted. The invention still further provides isolated PGC-1 proteins, fusion proteins, antigenic peptides and anti-PGC-1 antibodies. Diagnostic, screening, and therapeutic methods utilizing compositions of the invention are also provided.

RELATED APPLICATIONS

This application is a continuation application of U.S. Application Ser.No. 09/900,236, filed on Jul. 5, 2001, which is a divisional applicationof U.S. Application Ser. No. 09/203,453, filed on Dec. 1, 1998, now U.S.Pat. No. 6,426,411 allowed, which is a continuation-in-part of U.S.application Ser. No. 09/086,912, filed on May 29, 1998, now U.S. Pat.No. 6,166,192, which claims the benefit of U.S. Provisional ApplicationSer. No. 60/048,107, filed on May 30, 1997, the contents of all of whichare incorporated herein by reference.

GOVERNMENT SUPPORT

Work described herein was supported under grant 5R37DK31405 awarded bythe National Institutes of Health. The U.S. government therefore mayhave certain rights in this invention.

BACKGROUND OF THE INVENTION

Vertebrates possess two distinct types of adipose tissue: white adiposetissue (WAT) and brown adipose tissue (BAT). WAT stores and releases fataccording to the nutritional needs of the animal. BAT burns fat,releasing the energy as heat (i.e., nonshivering heat). The uniquethermogenic properties of BAT reflect the activities of specializedmitochondria that contain the brown adipocyte-specific gene productuncoupling protein (UCP). Sears, I. B. et al. (1996) Mol. Cell. Biol.16(7):3410-3419. UCP is a mitochondrial proton carrier that uncouplesrespiration from oxidative phosphorylation by collapsing the protongradient established from fatty acid oxidation without concomitant ATPsynthesis (Nicholls, D. and Locke, R. (1984) Physiol. Rev. 64:1-64).

UCP expression is tightly regulated, primarily by sympathetic nervoussystems, in response to physiological signals, such as cold exposure andexcess caloric intake (Girardier, L. and Seydoux, J. (1986) “NeuralControl of Brown Adipose Tissue” In P. Trayhurn and D. Nichols (eds.)Brown Adipose Tissue (Arnold, London, 1986) pp. 122-151. Norepinephrinereleased from the local neurons interacts with β-adrenergic receptors onthe brown adipocyte cell membrane, causing an increase in intracellularcyclic AMP (cAMP) levels (Sears, I. B. et al. (1996) Mol. Cell. Biol.16(7):3410-3419). An increased level of transcription of the UCP gene isa critical component in the cascade of events leading to elevated BATthermogenesis in response to increased cAMP (Kopecky, J. et al. (1990)J. Biol. Chem. 265:22204-22209; Rehnmark, S. M. et al. (1990) J. Biol.Chem. 265:16464-16471; Ricquier, D. F. et al. (1986) J. Biol. Chem.261:13905-13910). BAT thermogenesis is used both (1) to maintainhomeothermy by increasing thermogenesis in response to lowertemperatures and (2) to maintain energy balance by increasing energyexpenditure in response to increases in caloric intake (Sears, I. B. etal. (1996) Mol. Cell. Biol. 16(7):3410-3419). Nearly all experimentalrodent models of obesity are accompanied by diminished or defective BATfunction, usually as the first symptom in the progression of obesity(Himms-Hagen, J. (1989) Prog. Lipid Res. 28:67-115; Himms-Hagen, J.(1990) FASEB J. 4:2890-2898). In addition, ablation of BAT in transgenicmice by targeted expression of a toxin gene results in obesity (Lowell,B. et al. (1993) Nature 366:740-742). Thus, the growth anddifferentiation of brown adipocytes are key determinants in an animal'sability to maintain energy balance and prevent obesity (Sears, I. B. etal. (1996) Mol. Cell. Biol. 16(7):34100-3419).

Recently, several transcription factors have been identified whichpromote adipogenesis. These transcription factors include CCAAT/enhancerbinding protein (C/EBP) α, β, and δ and peroxisome proliferatoractivated receptor (PPAR)γ. See Spiegelman, B. M. and Flier, J. S.(1996) Cell 87:377-389 for a review. C/EBP family members such asC/EBPPα, β, and δ play important roles in the regulation ofadipocyte-specific gene expression. For example, C/EBPα cantransactivate the promoters of several genes expressed in the matureadipocyte (Herrera, R. et al. (1989) Mol. Cell. Biol. 9:5331-5339;Miller, S. G. et al. (1996) PNAS 93:5507-551; Christy, R. J. et al.(1989) Genes Dev. 3:1323-1335; Umek, R. M. et al. (1991) Science251:288-291; Kaestner, K. H. et al. (1990) PNAS 87:251-255; Delabrousse,F. C. et al. (1996) PNAS 93:4096-4101; Hwang, C. S. et al. (1996) PNAS93:873-877). Overexpression of C/EBP α can induce adipocytedifferentiation in fibroblasts (Freytag, S. O. et al. (1994) Genes Dev.8:1654-1663) whereas expression of antisense C/EBPα inhibits terminaldifferentiation of preadipocytes (Lin, F. T and Lane, M. D. (1992) GenesDev. 6:533-544). The physiological importance of C/EBPα was furtherdemonstrated by the generation of transgenic, C/EBPα-knockout mice.Although adipocytes are still present in these animals, they accumulatemuch less lipid and exhibit decreased adipocyte-specific gene expression(Wang, N. et al. (1995) Science 269:1108-1112). C/EBPα was found to havea synergistic relationship with another transcription factor, PPARγ, inpromoting adipocyte differentiation (See Brun, R. P. et al. (1996) Curr.Opin. Cell Biol. 8:826-832 for a review). PPARγ is a nuclear hormonereceptor which exists in two isoforms (γ1 and γ2) formed by alternativesplicing (Zhu, Y. et al. (1995) PNAS 92:7921-7925 ) and which appears tofunction as both a direct regulator of many fat-specific genes and alsoas a “master” regulator that can trigger the entire program ofadipogenesis (Spiegelman, B. M. and Flier, J. S. (1996) Cell87:377-389). PPARγ forms a heterodimer with RXRα and has been shown tobind directly to well characterized fat-specific enhancers from theadipocyte P2 (aP2: Tontonoz, P. (1994) Genes Dev. 8:1224-1234) andphosphoenolpyruvate carboxykinase (PEPCK) genes (Tontonoz, P. (1994)Mol. Cell. Biol. 15:351-357).

Although the UCP gene promoter includes binding sites for C/EBP (Yubero,P. et al. (1994) Biochem. Biophys. Res. Commun. 198:653-659) and aPPARγ-responsive element (Sears, I. B. et al. (1996) Mol. Cell. Biol.16(7):3410-3419), C/EBP and PPARγ do not seem to be sufficient to induceUCP expression (Sears, I. B. et al. (1996) Mol. Cell. Biol.16(7):3410-3419). It would be highly desirable, therefore, to identify apossible additional factor which acts in combination with either C/EBPor PPARγ to activate UCP expression and thus to promote BATthermogenesis.

SUMMARY OF THE INVENTION

This invention is based, at least in part, on the discovery of nucleicacid molecules which encode a family of novel molecules which can act incombination with PPARγ as a coactivator of UCP expression in BAT. Thesemolecules are referred to herein as PPARγ Coactivator 1 (“PGC-1”)proteins. Nucleic acid molecules encoding PGC-1 proteins are referred toherein as PGC-1 nucleic acid molecules. The PGC-1 molecules of theinvention are capable of, for example, modulating adipogenesis, e.g.,brown adipogenesis, and thermogenesis of a PGC-1 expressing tissue,e.g., BAT or muscle. Other functions of a PGC-1 family member of theinvention are described throughout the present application.

Accordingly, one aspect of the invention pertains to isolated nucleicacid molecules (e.g., cDNAs) comprising a nucleotide sequence encoding aPGC-1 protein or portions thereof (e.g., biologically active orantigenic portions), as well as nucleic acid fragments suitable asprimers or hybridization probes for the detection of PGC-1-encodingnucleic acid (e.g., mRNA). In particularly preferred embodiments, theisolated nucleic acid molecule comprises the nucleotide sequence of SEQID NO:1, SEQ ID NO:4 or a nucleotide sequence which is at least about50%, preferably at least about 60%, more preferably at least about 70%,yet more preferably at least about 80%, still more preferably at leastabout 90%, and most preferably at least about 95% or more homologous tothe nucleotide sequence of SEQ ID NO:1, SEQ ID NO:4, or the codingregion or a complement of either of these nucleotide sequences.

In other particularly preferred embodiments, the isolated nucleic acidmolecule of the invention comprises a nucleotide sequence whichhybridizes to or is at least about 50%, preferably at least about 60%,more preferably at least about 70%, yet more preferably at least about80%, still more preferably at least about 90%, and most preferably atleast about 95% or more homologous to the nucleotide sequence shown inSEQ ID NO:1, SEQ ID NO:4 or a portion (e.g., 400, 450, 500, or morenucleotides) of this nucleotide sequence.

In yet another preferred embodiment, the nucleic acid molecule includesa nucleotide sequence encoding a protein having the amino acid sequenceof SEQ ID NO: 2, SEQ ID NO:5. In yet another preferred embodiment, thenucleic acid molecule is at least 487 nucleotides in length. In anotherpreferred emnbodiment, the nucleic acid molecule comprises a fragment ofat least 487 nucleotides of the nucleotide sequence of SEQ ID NO:1, SEQID NO:4 or a complement thereof. In a further preferred embodiment, thenucleic acid molecule is at least 487 nucleotides in length and encodesa protein having an PGC-1 activity (as described herein).

Another embodiment of the invention features nucleic acid molecules,preferably PGC-1 nucleic acid molecules, which specifically detect PGC-1nucleic acid molecules relative to nucleic acid molecules encodingnon-PGC-1 proteins. For example, in one embodiment, such a nucleic acidmolecule is at least 350, 400, 450, or 487 nucleotides in length andhybridizes under stringent conditions to a nucleic acid moleculecomprising the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4, ora complement thereof. In a particularly preferred embodiment, thenucleic acid molecule comprises a fragment of at least 487 nucleotidesof the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:4, or a complementthereof. In preferred embodiments, the nucleic acid molecules are atleast 15 (e.g., contiguous) nucleotides in length and hybridize understringent conditions to nucleotides 10214, 316, 515-532, 895-1279,1427-1456, 2325-2387 of SEQ ID NO:1. In other preferred embodiments, thenucleic acid molecules include nucleotides 1-28, 50-232, 518-535,895-1219, 2325-2386, 2975-3023 of SEQ ID NO:4.

In other preferred embodiments, the isolated nucleic acid moleculeencodes the amino acid sequence of SEQ ID NO:2, SEQ ID NO:5 or an aminoacid sequence which is at least about 50%, preferably at least about60%, more preferably at least about 70%, yet more preferably at leastabout 80%, still more preferably at least about 90%, and most preferably95% or more homologous to the amino acid sequence of SEQ ID NO:2, SEQ IDNO:5. The preferred PGC-1 proteins of the present invention alsopreferably possess at least one of the PGC-1 biological activitiesdescribed herein.

In another embodiment, the isolated nucleic acid molecule encodes aprotein or portion thereof wherein the protein or portion thereofincludes an amino acid sequence which is sufficiently homologous to anamino acid sequence of SEQ ID NO:2, SEQ ID NO:5, e.g., sufficientlyhomologous to an amino acid sequence of SEQ ID NO:2, SEQ ID NO:5 suchthat the protein or portion thereof maintains a PGC-1 activity.Preferably, the protein or portion thereof encoded by the nucleic acidmolecule maintains one or more of the following biologicalactivities: 1) it can interact with (e.g., bind to) PPARγ; 2) it canmodulate PPARγ activity; 3) it can modulate UCP expression; 4) it canmodulate thermogenesis in adipocvtes, e.g., thermogenesis in brownadipocytes, or muscle; 5) it can modulate oxygen consumption inadipocytes or muscle; 6) it can modulate adipogenesis, e.g.,differentiation of white adipocytes into brown adipocytes; 7) it canmodulate insulin sensitivity of cells, e.g., insulin sensitivity ofmuscle cells, liver cells, adipocytes; 8) it can interact with (e.g.,bind to) nuclear hormone receptors, e.g., the thyroid hormone receptor,the estrogen receptor, the retinoic acid receptor; 9) it can modulatethe activity of nuclear hormone receptors; and 10) it can interact with(e.g., bind to) the transcription factor C/EBPα. In one embodiment, theprotein encoded by the nucleic acid molecule is at least about 50%,preferably at least about 60%, more preferably at least about 70%, yetmore preferably at least about 80%, still more preferably at least about90%, and most preferably at least about 95% or more homologous to theamino acid sequence of SEQ ID NO:2, SEQ ID NO:5 (e.g., the entire aminoacid sequence of SEQ ID NO:2, SEQ ID NO:5).

In yet another embodiment, the isolated nucleic acid molecule is derivedfrom a human and encodes a portion of a protein which includes one ormore of the following domains or motifs: a tyrosine phosphorylationsite, a cAMP phosphorylation site, a serine-arginine (SR) rich domain,an RNA binding motif, and an LXXLL (SEQ ID NO:3) motif which mediatesinteraction with a nuclear receptor. In another preferred embodiment,the isolated nucleic acid molecule is derived from a human and encodes aprotein (e.g., a PGC-1 fusion protein) which includes one or more of thedomains/motifs described herein and which has one or more of thefollowing biological activities: 1) it can interact with (e.g., bind to)PPARγ; 2) it can modulate PPARγ activity; 3) it can modulate UCPexpression; 4) it can modulate thermogenesis in adipocytes, e.g.,thermogenesis in brown adipocytes, or muscle; 5) it can modulate oxygenconsumption in adipocytes or muscle; 6) it can modulate adipogenesis,e.g., differentiation of white adipocytes into brown adipocytes; 7) itcan modulate insulin sensitivity of cells, e.g., insulin sensitivity ofmuscle cells, liver cells, adipocytes; 8) it can interact with (e.g.,bind to) nuclear hormone receptors, e.g., the thyroid hormone receptor,the estrogen receptor, the retinoic acid receptor; 9) it can modulatethe activity of nuclear hormone receptors; and 10) it can interact with(e.g., bind to) the transcription factor C/EBPα.

In another embodiment, the isolated nucleic acid molecule is at least 15nucleotides in length and hybridizes under stringent conditions to anucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1,SEQ ID NO:4 or to a nucleotide sequence which is at least about 50%,preferably at least about 60%, more preferably at least about 70%, yetmore preferably at least about 80%, still more preferably at least about90%, and most preferably at least about 95% or more homologous to thenucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4. Preferably, theisolated nucleic acid molecule corresponds to a naturally-occurringnucleic acid molecule. More preferably, the isolated nucleic acidencodes naturally-occurring human PGC-1 or a biologically active portionthereof. Moreover, given the disclosure herein of a PGC-1-encoding cDNAsequence (e.g., SEQ ID NO:1, SEQ ID NO:4), antisense nucleic acidmolecules (i.e., molecules which are complementary to the coding strandof the PGC-1 cDNA sequence) are also provided by the invention.

Another aspect of the invention pertains to vectors, e.g., recombinantexpression vectors, containing the nucleic acid molecules of theinvention and host cells into which such vectors have been introduced.In one embodiment, such a host cell is used to produce PGC-1 protein byculturing the host cell in a suitable medium. If desired, the PGC-1protein can be then isolated from the medium or the host cell.

Yet another aspect of the invention pertains to transgenic nonhumananimals in which a PGC-1 gene has been introduced or altered. In oneembodiment, the genome of the nonhuman animal has been altered byintroduction of a nucleic acid molecule of the invention encoding PGC-1as a transgene. In another embodiment, an endogenous PGC-1 gene withinthe genome of the nonhuman animal has been altered, e.g., functionallydisrupted, by homologous recombination.

Still another aspect of the invention pertains to an isolated PGC-1protein or a portion, e.g., a biologically active portion, thereof. In apreferred embodiment, the isolated PGC-1 protein or portion thereof canmodulate thermogenesis in BAT. In another preferred embodiment, theisolated PGC-1 protein or portion thereof is sufficiently homologous toan amino acid sequence of SEQ ID NO:2, SEQ ID NO:5 such that the proteinor portion thereof maintains one or more of the following biologicalactivities: 1) it can interact with (e.g., bind to) PPARγ; 2) it canmodulate PPARγ activity; 3) it can modulate UCP expression; 4) it canmodulate thermogenesis in adipocytes, e.g., thermogenesis in brownadipocytes, or muscle; 5) it can modulate oxygen consumption inadipocytes or muscle; 6) it can modulate adipogenesis, e.g.,differentiation of white adipocytes into brown adipocytes; 7) it canmodulate insulin sensitivity of cells, e.g., insulin sensitivity ofmuscle cells, liver cells, adipocytes; 8) it can interact with (e.g.,bind to) nuclear hormone receptors, e.g., the thyroid hormone receptor,the estrogen receptor, the retinoic acid receptor; 9) it can modulatethe activity of nuclear hormone receptors; and 10) it can interact with(e.g., bind to) the transcription factor C/EBPα.

In one embodiment, the biologically active portion of the PGC-1 proteinincludes a domain or motif, preferably a domain or motif which has aPGC-1 biological activity. The domain or motif can be a tyrosinephosphorylation site, a cAMP phosphorylation site, a serine-arginine(SR) rich domain, an RNA binding motif, and an LXXLL (SEQ ID NO:3) motifwhich mediates interaction with a nuclear receptor, or a combination ofone or more of these domains or motifs. Preferably, the biologicallyactive portion of the PGC-1 protein which includes one or more of thesedomains or motifs has one of the following biological activities: 1) itcan interact with (e.g., bind to) PPARγ; 2) it can modulate PPARγactivity; 3) it can modulate UCP expression; 4) it can modulatethermogenesis in adipocytes, e.g., thermogenesis in brown adipocytes, ormuscle; 5) it can modulate oxygen consumption in adipocytes or muscle;6) it can modulate adipogenesis, e.g., differentiation of whiteadipocytes into brown adipocytes; 7) it can modulate insulin sensitivityof cells, e.g., insulin sensitivity of muscle cells, liver cells,adipocytes; 8) it can interact with (e.g., bind to) nuclear hormonereceptors, e.g., the thyroid hormone receptor, the estrogen receptor,the retinoic acid receptor; 9) it can modulate the activity of nuclearhormone receptors; and 10) it can interact with (e.g., bind to) thetranscription factor C/EBPα.

The invention also provides an isolated preparation of a PGC-1 protein.In preferred embodiments, the PGC-1 protein comprises the amino acidsequence of SEQ ID NO:2, SEQ ID NO:5 or an amino acid sequence which isat least about 50%, preferably at least about 60%, more preferably atleast about 70%, yet more preferably at least about 80%, still morepreferably at least about 90%, and most preferably at least about 95% ormore homologous to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:5,e.g., the entire amino acid sequence of SEQ ID NO:2, SEQ ID NO:5. Inother embodiments, the isolated PGC-1 protein comprises an amino acidsequence which is at least about 50%, preferably at least about 60%,more preferably at least about 70%, yet more preferably at least about80%, still more preferably at least about 90%, and most preferably atleast about 95% or more homologous to the amino acid sequence of SEQ IDNO:2, SEQ ID NO:5 and has one or more of the PGC-1 biological activitiesdescribed herein. Alternatively, the isolated PGC-1 protein can comprisean amino acid sequence which is encoded by a nucleotide sequence whichhybridizes, e.g., hybridizes under stringent conditions, or is at leastabout 50%, preferably at least about 60%, more preferably at least about70%, yet more preferably at least about 80%, still more preferably atleast about 90%, and most preferably at least about 95% or morehomologous to the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:4. It isalso preferred that the preferred forms of PGC-1 also have one or moreof the PGC-1 biological activities described herein.

The PGC-1 protein (or polypeptide) or a biologically active portionthereof can be operatively linked to a non-PGC-1 polypeptide to form afusion protein. In addition, the PGC-1 protein or a biologically activeportion thereof can be incorporated into a pharmaceutical compositioncomprising the protein and a pharmaceutically acceptable carrier.

The PGC-1 protein of the invention, or portions or fragments thereof,can be used to prepare anti-PGC-1 antibodies. Accordingly, the inventionalso provides an antigenic peptide of PGC-1 which comprises at least 8amino acid residues of the amino acid sequence shown in SEQ ID NO:2, SEQID NO:5 (or an amino acid sequence which is at least about 50%homologous to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:5) andencompasses an epitope of PGC-1 such that an antibody raised against thepeptide forms a specific immune complex with PGC-1. Preferably, theantigenic peptide comprises at least 10 amino acid residues, morepreferably at least 15 amino acid residues, even more preferably atleast 20 amino acid residues, and most preferably at least 30 amino acidresidues. The invention further provides an antibody that specificallybinds PGC-1. In one embodiment, the antibody is monoclonal. In anotherembodiment, the antibody is coupled to a detectable substance. In yetanother embodiment, the antibody is incorporated into a pharmaceuticalcomposition comprising the antibody and a pharmaceutically acceptablecarrier.

Another aspect of the invention pertains to methods for modulating acell associated activity, e.g., proliferation, differentiation,survival, thermogenesis, oxygen consumption. Such methods includecontacting the cell with an agent which modulates PGC-1 protein activityor PGC-1 nucleic acid expression such that a cell associated activity isaltered relative to a cell associated activity (e.g., the same cellassociated activity) of the cell in the absence of the agent. In apreferred embodiment, the cell associated activity is thermogenesis andthe cell is a brown adipocyte. The agent which modulates PGC-1 activitycan be an agent which stimulates PGC-1 protein activity or PGC-1 nucleicacid expression. Examples of agents which stimulate PGC-1 proteinactivity or PGC-1 nucleic acid expression include small molecules,active PGC-1 proteins, and nucleic acids encoding PGC-1 that have beenintroduced into the cell. Examples of agents which inhibit PGC-1activity or expression include small molecules, antisense PGC-1 nucleicacid molecules, and antibodies that specifically bind to PGC-1. In apreferred embodiment, the cell is present within a subject and the agentis administered to the subject.

The present invention also pertains to methods for treating subjectshaving various disorders. For example, the invention pertains to methodsfor treating a subject having a disorder characterized by aberrant PGC-1protein activity or nucleic acid expression such as a weight disorder,e.g., obesity, anorexia, cachexia, or a disorder associated withinsufficient insulin activity, e.g., diabetes. These methods includeadministering to the subject a PGC-1 modulator (e.g., a small molecule)such that treatment of the subject occurs.

In one embodiment, the invention pertains to methods for treating asubject having a weight disorder, e.g., obesity, or a disorderassociated with insufficient insulin activity, e.g., diabetes,comprising administering to the subject a PGC-1 activator, e.g., a PGC-1protein or portion thereof or a compound or an agent thereby increasingthe expression or activity of PGC-1 such that treatment of the diseaseoccurs. Weight disorders, e.g., obesity, and disorders associated withinsufficient insulin activity can also be treated according to theinvention by administering to the subject having the disorder a PGC-1activator, e.g., a nucleic acid encoding a PGC-1 protein or portionthereof such that treatment occurs.

The invention also pertains to methods for detecting genetic lesions ina PGC-1 gene, thereby determining if a subject with the lesioned gene isat risk for (or is predisposed to have) a disorder characterized byaberrant or abnormal PGC-1 nucleic acid expression or PGC-1 proteinactivity, e.g., a weight disorder or a disorder associated withinsufficient insulin activity. In preferred embodiments, the methodsinclude detecting, in a sample of cells from the subject, the presenceor absence of a genetic lesion characterized by an alteration affectingthe integrity of a gene encoding a PGC-1 protein, or the misexpressionof the PGC-1 gene.

Another aspect of the invention pertains to methods for detecting thepresence of PGC-1 in a biological sample. In a preferred embodiment, themethods involve contacting a biological sample (e.g., a cardiomyocyte,hepatocyte, neuronal cell, a brown adipocyte or a muscle sample) with acompound or an agent capable of detecting PGC-1 protein or PGC-1 mRNAsuch that the presence of PGC-1 is detected in the biological sample.The compound or agent can be, for example, a labeled or labelablenucleic acid probe capable of hybridizing to PGC-1 mRNA or a labeled orlabelable antibody capable of binding to PGC-1 protein. The inventionfurther provides methods for diagnosis of a subject with, for example, aweight disorder or a disorder associated with insufficient insulinactivity, based on detection of PGC-1 protein or mRNA. In oneembodiment, the method involves contacting a cell or tissue sample(e.g., a brown adipocyte sample) from the subject with an agent capableof detecting PGC-1 protein or mRNA, determining the amount of PGC-1protein or mRNA expressed in the cell or tissue sample, comparing theamount of PGC-1 protein or mRNA expressed in the cell or tissue sampleto a control sample and forming a diagnosis based on the amount of PGC-1protein or mRNA expressed in the cell or tissue sample as compared tothe control sample. Preferably, the cell sample is a brown adipocytesample. Kits for detecting PGC-1 in a biological sample are also withinthe scope of the invention.

Still another aspect of the invention pertains to methods, e.g.,screening assays, for identifying a compound for treating a disordercharacterized by aberrant PGC-1 nucleic acid expression or proteinactivity, e.g., a weight disorder or a disorder associated withinsufficient insulin activity. These methods typically include assayingthe ability of the compound or agent to modulate the expression of thePGC-1 gene or the activity of the PGC-1 protein thereby identifying acompound for treating a disorder characterized by aberrant PGC-1 nucleicacid expression or protein activity. In a preferred embodiment, themethod involves contacting a biological sample, e.g., a cell or tissuesample, e.g., a brown adipocyte sample, obtained from a subject havingthe disorder with the compound or agent, determining the amount of PGC-1protein expressed and/or measuring the activity of the PGC-1 protein inthe biological sample, comparing the amount of PGC-1 protein expressedin the biological sample and/or the measurable PGC-1 biological activityin the cell to that of a control sample. An alteration in the amount ofPGC-1 nucleic acid expression or PGC-1 protein activity in the cellexposed to the compound or agent in comparison to the control isindicative of a modulation of PGC-1 nucleic acid expression and/or PGC-1protein activity.

The invention also pertains to methods for identifying a compound oragent which interacts with (e.g., binds to) a PGC-1 protein. Thesemethods include the steps of contacting the PGC-1 protein with thecompound or agent under conditions which allow binding of the compoundto the PGC-1 protein to form a complex and detecting the formation of acomplex of the PGC-1 protein and the compound in which the ability ofthe compound to bind to the PGC-1 protein is indicated by the presenceof the compound in the complex.

The invention further pertains to methods for identifying a compound oragent which modulates, e.g., stimulates or inhibits, the interaction ofthe PGC-1 protein with a target molecule, e.g., PPARγ, C/EBPα, a nuclearhormone receptor, e.g., the thyroid hormone receptor, the estrogenreceptor, the retinoic acid receptor. In these methods, the PGC-1protein is contacted, in the presence of the compound or agent, with thetarget molecule under conditions which allow binding of the targetmolecule to the PGC-1 protein to form a complex. An alteration, e.g., anincrease or decrease, in complex formation between the PGC-1 protein andthe target molecule as compared to the amount of complex formed in theabsence of the compound or agent is indicative of the ability of thecompound or agent to modulate the interaction of the PGC-1 protein witha target molecule.

BRIEF DESCRIPTION OF THE DRAWING

FIGS. 1A-1C depict the mouse PGC-1 nucleotide (SEQ ID NO:1) and aminoacid (SEQ ID NO:2) sequence.

FIGS. 2A-2B depict an analysis of the mouse PGC-1 sequence. Thefollowing domains are underlined in FIG. 2A: SR domains (amino acids565-598 and 617-631), an RNA-binding domain (amino acid 677-709), threeconsensus sites for phosphorylating protein kinase A (amino acids238-241, 373-376 and 655-668), and an LXXLL (SEQ ID NO:3) motif (aminoacids 142-146).

FIG. 2B is a schematic representation of the structure of mouse PGC-1.Arrows indicate putative protein kinase A phosphorylation sites havingthe consensus sequence (R, K)2x(ST). The gray box indicates the SR richregion domain and black box indicates the RNA-binding domain.

FIGS. 3A-3B are bar graphs depicting the effect of mouse PGC-1 instimulating the transactivation of the UCP-1 promoter by PPARγ and thethyroid hormone receptor (TR). FIG. 3A depicts the increasedtranscription activation of the CAT reporter gene under the control ofthe UCP-1 promoter with respect to the indicated ligands/hormones inRAT1 IR cells. FIG. 3B is a graph depicting the increased transcriptionactivation of a reporter CAT gene under the control of UAS sequences(five copies) using mouse PGC-1 linked to GAL4 DBD.

FIG. 4 is a diagram of different mouse PGC-1 deletions to identify thedomain of PGC-1 which interacts with PPARγ. Indicated in the Figure areschematic representations of the PGC-1 deletions with the correspondingpercentage of input material that bound to PPARγ. The LXXLL (SEQ IDNO:3) motif is located at amino acid residues 142-146. The black boxcorresponds to the PPARγ-binding domain of PGC-1 (amino acid 292-338).

FIG. 5 is a diagram of different mouse PPARγ deletions to identify thedomain of PPARγ which interacts with PGC-1. Indicated in the Figure areschematic representations of the PPARγ deletions with the correspondingpercentage of input binding to PGC-1.

FIG. 6 is a bar graph depicting the effect in oxygen consumption ofchronic treatment of PGC-1 infected and control cells with cAMP andRetinoic Acid (RA).

FIGS. 7A-7B depict the human PGC-1 nucleotide (SEQ ID NO:4) sequence.

FIG. 8 depicts the human PGC-1 amino acid (SEQ ID NO:5) sequence.

FIGS. 9A-9C depict an alignment between the human PGC-1 amino acidsequence (SEQ ID NO:5) and the mouse PGC-1 amino acid sequence (SEQ IDNO:2). This alignment was performed with BLAST software found at theNational Center for Biotechnology Information (NCBI) web site (Altschul,S. F. et al. (1990) J. Mol. Biol. 215:403-410; Madden, T. L. et al.(1996) Methods Enzymol. 266:131-141) and it was determined that humanPGC-1 has a 94% identity to mouse PGC-1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery of novel molecules,referred to herein as PGC-1 nucleic acid and protein molecules, whichcomprise a family of molecules having certain conserved structural andfunctional features, and which play a role in or function in adipocyteassociated activities. The term “family” when referring to the proteinand nucleic acid molecules of the invention is intended to mean two ormore proteins or nucleic acid molecules having a common structuraldomain and having sufficient amino acid or nucleotide sequence homologyas defined herein. Such family members can be naturally occurring andcan be from either the same or different species. For example, a familycan contain a first protein of human origin, as well as other, distinctproteins of human origin or alternatively, can contain homologues ofnon-human origin. Members of a family may also have common functionalcharacteristics.

In one embodiment, a PGC-1 molecule can modulate adipogenesis, e.g.,adipogenesis of brown adipocytes and muscle cells. In anotherembodiment, a PGC-1 molecule can modulate thermogenesis in brownadipocytes. For example, a PGC-1 molecule of the invention can increasethermogenesis in adipocytes of an individual, thereby promoting weightloss in the individual. Thus, a PGC-1 molecule of the invention can beused to treat obesity. Additionally, the increase in thermogenicactivity caused by a PGC-1 molecule can also increase insulinsensitivity of the adipocytes as well as of muscle cells and livercells. Thus, a PGC-1 molecule of the invention can also be used to treatdisorders characterized by insufficient insulin activity such asdiabetes. Alternatively, inhibition of the activity of a PGC-1 moleculeof the invention can decrease thermogenesis in adipocytes of anindividual, thereby inhibiting weight loss in the individual. Thus, themodulators of PGC-1 molecules of the invention can be used to treatundesirable weight loss, e.g., cachexia, anorexia. Moreover, a PGC-1molecule of the invention can also be used as targets to screenmolecules, e.g., small molecules, which can modulate PGC-1 activity.PGC-1 molecule modulators can also be used to treat weight disorders,e.g., cachexia, anorexia, obesity, or disorders characterized byinsufficient insulin activity.

Mouse PGC-1 nucleic acid molecules were identified from mouse brownadipocytes based on their ability, as determined using a yeast twohybrid assay (described in Example I) to bind to PPARγ. As describedabove, PPARγ is a nuclear hormone receptor which functions as both adirect regulator of many fat-specific genes and also as a “master”regulator that can trigger the entire program of adipogenesis. Moreover,as the UCP gene promoter includes a PPARγ-responsive element, amodulator of PPARγ can modulate adipogenesis and UCP expression. UCPexpression can result in thermogenesis.

The nucleotide sequence of the mouse and human PGC-1 cDNA and thepredicted amino acid sequence of the mouse and human PGC-1 proteins areshown in FIGS. 1A, 1B, 1C, 2A, 7A, 7B, and 8, and in SEQ ID NOs:1, 2, 4,and 5, respectively. Using all or a portion of the mouse nucleotidesequence (e.g., a 5′ portion of SEQ ID NO:1, e.g., nucleotides 1-50 ofSEQ ID NO:1) to probe a cDNA library from a human cell line such as ahuman muscle, heart, kidney, or brain cell line, the human PGC-1nucleotide sequence was obtained using routine experimentation asdescribed in Example II. The mouse PGC-1 gene, which is approximately3066 nucleotides in length, encodes a full length protein having amolecular weight of approximately 120 kD and which is approximately 797amino acid residues in length. The human PGC-1 gene, which isapproximately 3023 nucleotides in length, encodes a full length proteinhaving a molecular weight of approximately 120 kD and which isapproximately 798 amino acid residues in length. PGC-1 family memberproteins include several domains/motifs. These domains/motifs include:two putative tyrosine phosphorylation sites (amino acid residues 204-212and 378-385 of SEQ ID NO:2, and amino acid residues 205-213 and 379-386of SEQ ID NO:5), three putative cAMP phosphorylation sites (amino acidresidues 238-241, 373-376, and 655-658 of SEQ ID NO:2, and 239-242,374-377, and 656-658 of SEQ ID NO:5), a serine-arginine (SR) rich domain(amino acid residues 562-600 of SEQ ID NO:2, and 563-601 of SEQ IDNO:5), an RNA binding motif (amino acid residues 656-709 of SEQ ID NO:2,and 657-710 of SEQ ID NO:5), and an LXXLL motif (amino acids 142-146 ofSEQ ID NO:2, 143-147 of SEQ ID NO:5; SEQ ID NO:3) which mediatesinteraction with a nuclear receptor. As used herein, a tyrosinephosphorylation site is an amino acid sequence which includes at leastone tyrosine residue which can be phosphorylated by a tyrosine proteinkinase. Typically, a tyrosine phosphorylation site is characterized by alysine or an arginine about seven residues to the N-terminal side of thephosphorylated tyrosine. An acidic residue (asparagine or glutamine) isoften found at either three or four residues to the N-terminal side ofthe tyrosine (Patschinsky, T. et al. (1982) Proc. Natl. Acad. Sci. USA79:973-977); Hunter, T. (1982) J. Biol. Chem. 257:4843-4848; Cooper, J.A. et al. (1984) J. Biol. Chem. 259:7835-7841). As used herein, a cAMPphosphorylation site is an amino acid sequence which includes a serineor threonine residue which can be phosphorylated by a cAMP-dependentprotein kinase. Typically, the cAMP phosphorylation site ischaracterized by at least two consecutive basic residues to theN-terminal side of the serine or threonine (Fremisco, J. R. et al.(1980) J. Biol. Chem. 255:4240-4245; Glass, D. B. and Smith, S. B.(1983) J. Biol. Chem. 258:14797-14803; Glass, D. B. et al. (1986) J.Biol. Chem. 261:2987-2993). As used herein, a serine-arginine richdomain is an amino acid sequence which is rich in serine and arginineresidues. Typically, SR rich domains are domains which interact with theCTD domain of RNA polymerase II or are involved in splicing functions.As used herein, an RNA binding motif is an amino acid sequence which canbind an RNA molecule or a single stranded DNA molecule. RNA bindingmotifs are described in Lodish, H., Darnell, J., and Baltimore, D.Molecular Cell Biology, 3rd ed (W. H. Freeman and Company, New York,N.Y., 1995). As used herein, an LXXLL (SEQ ID NO:3) refers to a motifwherein X can be any amino acid and which mediates an interactionbetween an nuclear receptor and a coactivator (Heery et al. (1997)Nature 397:733-736; Torchia et al. (1997) Nature 387:677-684).

The PGC-1 protein is expressed in muscle, heart, kidney, brain and brownadipose tissue but not in white adipose tissue. In tissue from coldacclimated animals, PGC-1 expression was highly induced in brown adiposetissue. Moreover, in tissue from cold acclimated animals, PGC-1expression was brown adipose tissue specific. PGC-1 expression intissues from cold acclimated animals parallels expression of UCP, thebrown adipose tissue marker responsible for the thermogenic activity ofthis tissue.

The PGC-1 protein or a biologically active portion or fragment of theinvention can have one or more of the following activities: 1) it caninteract with (e.g., bind to) PPARγ; 2) it can modulate PPARγ activity;3) it can modulate UCP expression; 4) it can modulate thermogenesis inadipocytes, e.g., thermogenesis in brown adipocytes, or muscle; 5) itcan modulate oxygen consumption in adipocytes or muscle; 6) it canmodulate adipogenesis, e.g., differentiation of white adipocytes intobrown adipocytes; 7) it can modulate insulin sensitivity of cells, e.g.,insulin sensitivity of muscle cells, liver cells, adipocytes; 8) it caninteract with (e.g., bind to) nuclear hormone receptors, e.g., thethyroid hormone receptor, the estrogen receptor, the retinoic acidreceptor; 9) it can modulate the activity of nuclear hormone receptors;and 10) it can interact with (e.g., bind to) the transcription factorC/EBPα.

Various aspects of the invention are described in further detail in thefollowing subsections:

I. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode PGC-1 or biologically active portions thereof, as well asnucleic acid fragments sufficient for use as hybridization probes toidentify PGC-1-encoding nucleic acid (e.g., PGC-1 mRNA). As used herein,the term “nucleic acid molecule” is intended to include DNA molecules(e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogsof the DNA or RNA generated using nucleotide analogs. The nucleic acidmolecule can be single-stranded or double-stranded, but preferably isdouble-stranded DNA. An “isolated” nucleic acid molecule is one which isseparated from other nucleic acid molecules which are present in thenatural source of the nucleic acid. Preferably, an “isolated” nucleicacid is free of sequences which naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forexample, in various embodiments, the isolated PGC-1 nucleic acidmolecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5kb or 0.1 kb of nucleotide sequences which naturally flank the nucleicacid molecule in genomic DNA of the cell from which the nucleic acid isderived (e.g., a brown adipocyte). Moreover, an “isolated” nucleic acidmolecule, such as a cDNA molecule, can be substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or chemical precursors or other chemicals when chemicallysynthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:4 or anucleotide sequence which is at least about 50%, preferably at leastabout 60%, more preferably at least about 70%, yet more preferably atleast about 80%, still more preferably at least about 90%, and mostpreferably at least about 95% or more homologous to the nucleotidesequence shown in SEQ ID NO:1, SEQ ID NO:4 or a portion thereof (e.g.,400, 450, 500, or more nucleotides), can be isolated using standardmolecular biology techniques and the sequence information providedherein. For example, a human PGC-1 cDNA can be isolated from a humanheart, kidney,. or brain cell line (from Stratagene, LaJolla, Calif., orClontech, Palo Alto, Calif.) using all or portion of SEQ ID NO:1, SEQ IDNO:4 as a hybridization probe and standard hybridization techniques(e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T.Molecular Cloning: A Laboratory Manual. 2nd, ed, Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989). Moreover, a nucleic acid molecule encompassing all or aportion of SEQ ID NO:1, SEQ ID NO:4 or a nucleotide sequence which is atleast about 50%, preferably at least about 60%, more preferably at leastabout 70%, yet more preferably at least about 80%, still more preferablyat least about 90%, and most preferably at least about 95% or morehomologous to the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4can be isolated by the polymerase chain reaction using oligonucleotideprimers designed based upon the sequence of SEQ ID NO:1, SEQ ID NO:4 orthe homologous nucleotide sequence. For example, mRNA can be isolatedfrom heart cells, kidney cells, brain cells, or brown adipocytes (e.g.,by the guanidinium-thiocyanate extraction procedure of Chirgwin et al.(1979) Biochemistry 18: 5294-5299) and cDNA can be prepared usingreverse transcriptase (e.g., Moloney MLV reverse transcriptase,available from Gibco/BRL, Bethesda, Md.; or AMV reverse transcriptase,available from Seikagaku America, Inc., St. Petersburg, Fla.). Syntheticoligonucleotide primers for PCR amplification can be designed based uponthe nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4 or to thehomologous nucleotide sequence. A nucleic acid of the invention can beamplified using cDNA or, alternatively, genomic DNA, as a template andappropriate oligonucleotide primers according to standard PCRamplification techniques. The nucleic acid so amplified can be clonedinto an appropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to a PGC-1 nucleotidesequence can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid molecule of theinvention comprises the nucleotide sequence shown in SEQ ID NO:1, SEQ IDNO:4 or a nucleotide sequence which is at least about 50%, preferably atleast about 60%, more preferably at least about 70%, yet more preferablyat least about 80%, still more preferably at least about 90%, and mostpreferably at least about 95% or more homologous to the nucleotidesequence shown in SEQ ID NO:1, SEQ ID NO:4. The sequence of SEQ ID NO:1corresponds to the mouse PGC-1 cDNA. This cDNA comprises sequencesencoding the PGC-1 protein (i.e., “the coding region”, from nucleotides92 to 2482), as well as 5′ untranslated sequences (nucleotides 1 to 91 )and 3′ untranslated sequences (nucleotides 2483 to 3066). Alternatively,the nucleic acid molecule can comprise only the coding region of SEQ IDNO:1 (e.g., nucleotides 92 to 2482) or the homologous nucleotidesequence. The sequence of SEQ ID NO:4 corresponds to the human PGC-1cDNA. This cDNA comprises sequences encoding the PGC-1 protein (i.e.,“the coding region”, from nucleotides 89 to 2482), as well as 5′untranslated sequences (nucleotides 1 to 88) and 3′ untranslatedsequences (nucleotides 2513 to 3023). Alternatively, the nucleic acidmolecule can comprise only the coding region of SEQ ID NO:4 (e.g.,nucleotides 89 to 2482) or the homologous nucleotide sequence.

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule which is a complement ofthe nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4 or anucleotide sequence which is at least about 50%, preferably at leastabout 60%, more preferably at least about 70%, yet more preferably atleast about 80%, still more preferably at least about 90%, and mostpreferably at least about 95% or more homologous to the nucleotidesequence shown in SEQ ID NO:1, SEQ ID NO:4. A nucleic acid moleculewhich is complementary to the nucleotide sequence shown in SEQ ID NO:1,SEQ ID NO:4 or to a nucleotide sequence which is at least about 50%,preferably at least about 60%, more preferably at least about 70%, yetmore preferably at least about 80%, still more preferably at least about90%, and most preferably at least about 95% or more homologous to thenucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4 is one which issufficiently complementary to the nucleotide sequence shown in SEQ IDNO:1, SEQ ID NO:4 or to the homologous sequence such that it canhybridize to the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4or to the homologous sequence, thereby forming a stable duplex.

In still another preferred embodiment, an isolated nucleic acid moleculeof the invention comprises a nucleotide sequence which is at least about50%, preferably at least about 60%, more preferably at least about 70%,yet more preferably at least about 80%, still more preferably at leastabout 90%, and most preferably at least about 95% or more homologous tothe nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4 or a portionof this nucleotide sequence. In an additional preferred embodiment, anisolated nucleic acid molecule of the invention comprises a nucleotidesequence which hybridizes, e.g., hybridizes under stringent conditions,to the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4 or to anucleotide sequence which is at least about 50%, preferably at leastabout 60%, more preferably at least about 70%, yet more preferably atleast about 80%, still more preferably at least about 90%, and mostpreferably at least about 95% or more homologous to the nucleotidesequence shown in SEQ ID NO:1, SEQ ID NO:4.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the coding region of SEQ ID NO:1, SEQ ID NO:4 or the codingregion of a nucleotide sequence which is at least about 50%, preferablyat least about 60%, more preferably at least about 70%, yet morepreferably at least about 80%, still more preferably at least about 90%,and most preferably at least about 95% or more homologous to thenucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4, for example afragment which can be used as a probe or primer or a fragment encoding abiologically active portion of PGC-1. The nucleotide sequence determinedfrom the cloning of the PGC-1 gene from a mouse allows for thegeneration of probes and primers designed for use in identifying and/orcloning other PGC-1 family members, as well as PGC-1 homologues in othercell types, e.g. from other tissues, as well as PGC-1 homologues fromother mammals such as humans. The probe/primer typically comprisessubstantially purified oligonucleotide. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, preferably at least about 25,more preferably about 40, 50 or 75 consecutive nucleotides of SEQ IDNO:1, SEQ ID NO:4 sense, an anti-sense sequence of SEQ ID NO:1, SEQ IDNO:4, or naturally occurring mutants thereof. Primers based on thenucleotide sequence in SEQ ID NO:1, SEQ ID NO:4 can be used in PCRreactions to clone PGC-1 homologues.

In an exemplary embodiment, a nucleic acid molecule of the presentinvention comprises a nucleotide sequence which is about 100, preferably100-200, preferably 200-300, more preferably 300-400, and even morepreferably 400-487 nucleotides in length and hybridizes under stringenthybridization conditions to a nucleic acid molecule of SEQ ID NO:1, SEQID NO:4.

Probes based on the PGC-1 nucleotide sequences can be used to detecttranscripts or genomic sequences encoding the same or homologousproteins. In preferred embodiments, the probe further comprises a labelgroup attached thereto, e.g. the label group can be a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. Such probes canbe used as a part of a diagnostic test kit for identifying cells ortissue which misexpress a PGC-1 protein, such as by measuring a level ofa PGC-1-encoding nucleic acid in a sample of cells from a subject e.g.,detecting PGC-1 mRNA levels or determining whether a genomic PGC-1 genehas been mutated or deleted.

In one embodiment, the nucleic acid molecule of the invention encodes aprotein or portion thereof which includes an amino acid sequence whichis sufficiently homologous to an amino acid sequence of SEQ ID NO:2, SEQID NO:5 such that the protein or portion thereof maintains one or moreof the following biological activities: 1) it can interact with (e.g.,bind to) PPARγ; 2) it can modulate PPARγ activity; 3) it can modulateUCP expression; 4) it can modulate thermogenesis in adipocytes, e.g.,thermogenesis in brown adipocytes, or muscle; 5) it can modulate oxygenconsumption in adipocytes or muscle; 6) it can modulate adipogenesis,e.g., differentiation of white adipocytes into brown adipocytes; 7) itcan modulate insulin sensitivity of cells, e.g., insulin sensitivity ofmuscle cells, liver cells, adipocytes; 8) it can interact with (e.g.,bind to) nuclear hormone receptors, e.g., the thyroid hormone receptor,the estrogen receptor, the retinoic acid receptor; 9) it can modulatethe activity of nuclear hormone receptors; and 10) it can interact with(e.g., bind to) the transcription factor C/EBPα.

As used herein, the language “sufficiently homologous” refers toproteins or portions thereof which have amino acid sequences whichinclude a minimum number of identical or equivalent (e.g., an amino acidresidue which has a similar side chain as an amino acid residue in SEQID NO:2, SEQ ID NO:5) amino acid residues to an amino acid sequence ofSEQ ID NO:2, SEQ ID NO:5 such that the protein or portion thereofmaintains one or more of the following biological activities: 1) it caninteract with (e.g., bind to) PPARγ; 2) it can modulate PPARγ activity;3) it can modulate UCP expression; 4) it can modulate thermogenesis inadipocytes, e.g., thermogenesis in brown adipocytes, or muscle; 5) itcan modulate oxygen consumption in adipocytes or muscle; 6) it canmodulate adipogenesis, e.g., differentiation of white adipocytes intobrown adipocytes; 7) it can modulate insulin sensitivity of cells, e.g.,insulin sensitivity of muscle cells, liver cells, adipocytes; 8) it caninteract with (e.g., bind to) nuclear hormone receptors, e.g., thethyroid hormone receptor, the estrogen receptor, the retinoic acidreceptor; 9) it can modulate the activity of nuclear hormone receptors;and 10) it can interact with (e.g., bind to) the transcription factorC/EBPα. In another embodiment, the protein is at least about 50%,preferably at least about 60%, more preferably at least about 70%, yetmore preferably at least about 80%, still more preferably at least about90%, and most preferably at least about 95% or more homologous to theentire amino acid sequence of SEQ ID NO:2, SEQ ID NO:5.

Portions of proteins encoded by the PGC-1 nucleic acid molecule of theinvention are preferably biologically active portions of the PGC-1protein. As used herein, the term “biologically active portion of PGC-1”is intended to include a portion, e.g., a domain/motif, of PGC-1 thathas one or more of the following activities: 1) it can interact with(e.g., bind to) PPARγ; 2) it can modulate PPARγ activity; 3) it canmodulate UCP expression; 4) it can modulate thermgenesis in adipocytes,e.g., thermogenesis in brown adipocytes, or muscle; 5) it can modulateoxygen consumption in adipocytes or muscle; 6) it can modulateadipogenesis, e.g., differentiation of white adipocytes into brownadipocytes; 7) it can modulate insulin sensitivity of cells, e.g.,insulin sensitivity of muscle cells, liver cells, adipocytes; 8) it caninteract with (e.g., bind to) nuclear hormone receptors, e.g., thethyroid hormone receptor, the estrogen receptor, the retinoic acidreceptor; 9) it can modulate the activity of nuclear hormone receptors;and 10) it can interact with (e.g., bind to) the transcription factorC/EBPα.

Standard binding assays, e.g., immunoprecipitations and yeast two-hybridassays as described herein, can be performed to determine the ability ofa PGC-1 protein or a biologically active portion thereof to interactwith (e.g., bind to) PPARγ, C/EBPPα, and nuclear hormone receptors. If aPGC-1 family member is found to interact with PPARγ, C/EBPα, and/ornuclear hormone receptors, then they are also likely to be modulators ofthe activity of PPARγ, C/EBPPα, and nuclear hormone receptors.

To determine whether a PGC-1 family member of the present inventionmodulates UCP expression, in vitro transcriptional assays can beperformed. To perform such an assay, the full length promoter andenhancer of UCP can be linked to a reporter gene such as chloramphenicolacetyltransferase (CAT) and introduced into host cells. The same hostcells can then be transfected with PPARγ/RXRα and nucleic acid encodingthe PGC-1 molecule. The effect of the PGC-1 molecule can be measured bytesting CAT activity and comparing it to CAT activity in cells which donot contain nucleic acid encoding the PGC-1 molecule. An increase ordecrease in CAT activity indicates a modulation of UCP expression andsince UCP expression is known to be a critical component in the cascadeof events leading to elevated thermogenesis, this assay can also measurethe ability of the PGC-1 molecule to modulate thermogenesis inadipocytes.

The above described assay for testing the ability of a PGC-1 molecule tomodulate UCP expression can also be used to test the ability of thePGC-1 molecule to modulate adipogenesis, e.g., differentiation of whiteadipose tissue to brown adipose tissue, as UCP expression is specific tobrown adipose tissue. If a PGC-1 molecule can modulate UCP expression iscan most likely modulate the differentiation of white adipose tissue tobrown adipose tissue. Alternatively, the ability of a PGC-1 molecule tomodulate the differentiation of white adipose tissue to brown adiposetissue can be measured by introducing a PGC-1 molecule into a cell,e.g., a white adipocyte, and measuring the number of mitochondria in thecell as compared to the number of mitochondria in a control cell whichdoes not contain the PGC-1 molecule. As brown adipocytes are known tocontain substantially greater numbers of mitochondria than whiteadipocytes, an increase or decrease in the number of mitochondria (or ina mitochondrial marker such as cytochrome c oxidase) in the test cell ascompared to the control cell indicates that the PGC-1 molecule canmodulate differentiation of white adipose tissue to brown adiposetissue.

The ability of a PGC-1 molecule to modulate insulin sensitivity of acell can be determined by performing an assay in which cells, e.g.,muscle cells, liver cells, or adipocytes, are transformed to express thePGC-1 protein, incubated with radioactively labeled glucose (¹⁴Cglucose), and treated with insulin. An increase or decrease in glucosein the cells containing PGC-1 as compared to the control cells indicatesthat the PGC-1 can modulate insulin sensitivity of the cells.Alternatively, the cells containing PGC-1 can be incubated with aradioactively labeled phosphate source (e.g., [³²P]ATP) and treated withinsulin. Phosphorylation of proteins in the insulin pathway, e.g.,insulin receptor, can then be measured. An increase or decrease inphosphorylation of a protein in the insulin pathway in cells containingPGC-1 as compared to the control cells indicates that the PGC-1-canmodulate insulin sensitivity of the cells.

In one embodiment, the biologically active portion of PGC-1 comprises adomain or motif. Examples of such domains/motifs include a tyrosinephosphorylation site, a cAMP phosphorylation site, a serine-arginine(SR) rich domain, an RNA binding motif, and an LXXLL (SEQ ID NO:3) motifwhich mediates interaction with a nuclear receptor. In a preferredembodiment, the biologically active portion of the protein whichincludes the domain or motif can modulate differentiation of whiteadipocytes to brown adipocytes and/or thermogenesis in brown adipocytes.These domains are described in detail herein. Additional nucleic acidfragments encoding biologically active portions of PGC-1 can be preparedby isolating a portion of SEQ ID NO:1, SEQ ID NO:4 or a homologousnucleotide sequence, expressing the encoded portion of PGC-1 protein orpeptide (e.g., by recombinant expression in vitro) and assessing theactivity of the encoded portion of PGC-1 protein or peptide.

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4 (andportions thereof) due to degeneracy of the genetic code and thus encodethe same PGC-1 protein as that encoded by the nucleotide sequence shownin SEQ ID NO:1, SEQ ID NO:4. In another embodiment, an isolated nucleicacid molecule of the invention has a nucleotide sequence encoding aprotein having an amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:5or a protein having an amino acid sequence which is at least about 50%,preferably at least about 60%, more preferably at least about 70%, yetmore preferably at least about 80%, still more preferably at least about90%, and most preferably at least about 95% or more homologous to theamino acid sequence of SEQ ID NO:2, SEQ ID NO:5.

In addition to the mouse and human PGC-1 nucleotide sequences shown inSEQ ID NO:1 and SEQ ID NO:4, it will be appreciated by those skilled inthe art that DNA sequence polymorphisms that lead to changes in theamino acid sequences of PGC-1 may exist within a population (e.g., amammalian population, e.g., a human population). Such geneticpolymorphism in the PGC-1 gene may exist among individuals within apopulation due to natural allelic variation. As used herein, the terms“gene” and “recombinant gene” refer to nucleic acid molecules comprisingan open reading frame encoding a PGC-1 protein, preferably a mammalian,e.g., human, PGC-1 protein. Such natural allelic variations cantypically result in 1-5% variance in the nucleotide sequence of thePGC-1 gene. Any and all such nucleotide variations and resulting aminoacid polymorphisms in PGC-1 that are the result of natural allelicvariation and that do not alter the functional activity of PGC-1 areintended to be within the scope of the invention. Moreover, nucleic acidmolecules encoding PGC-1 proteins from other species, and thus whichhave a nucleotide sequence which differs from the mouse sequence of SEQID NO:1, SEQ ID NO:4, are intended to be within the scope of theinvention. Nucleic acid molecules corresponding to natural allelicvariants and human homologues of the mouse PGC-1 cDNA of the inventioncan be isolated based on their homology to the mouse PGC-1 nucleic aciddisclosed herein using the mouse cDNA, or a portion thereof, as ahybridization probe according to standard hybridization techniques understringent hybridization conditions (see Example II).

Moreover, nucleic acid molecules encoding other PGC-1 family members andthus which have a nucleotide sequence which differs from the PGC-1sequences of SEQ ID NO:1 or SEQ ID NO:4 are intended to be within thescope of the invention. For example, a PGC-2 cDNA can be identifiedbased on the nucleotide sequence of human PGC-1 or mouse PGC-1.Moreover, nucleic acid molecules encoding PGC-1 proteins from differentspecies, and thus which have a nucleotide sequence which differs fromthe PGC-1 sequences of SEQ ID NO:1 or SEQ ID NO:4 are intended to bewithin the scope of the invention. For example, rat PGC-1 cDNA can beidentified based on the nucleotide sequence of a human PGC-1.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 15 nucleotides in length and hybridizes understringent conditions to the nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:1, SEQ ID NO:4 or a nucleotide sequencewhich is about 60%, preferably at least about 70%, more preferably atleast about 80%, still more preferably at least about 90%, and mostpreferably at least about 95% or more homologous to the nucleotidesequence of SEQ ID NO:1, SEQ ID NO:4. In other embodiments, the nucleicacid is at least 30, 50, 100, 250 or 500 nucleotides in length. As usedherein, the term “hybridizes under stringent conditions” is intended todescribe conditions for hybridization and washing under which nucleotidesequences at least 60% homologous to each other typically remainhibridized to each other. Preferably, the conditions are such thatsequences at least about 65%, more preferably at least about 70%, andeven more preferably at least about 75% or more homologous to each othertypically remain hybridized to each other. Such stringent conditions areknown to those skilled in the art and can be found in Current Protocolsin Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Apreferred, non-limiting example of stringent hybridization conditionsare hybridization in 6X sodium chloride/sodium citrate (SSC) at about45° C., followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65°C. Preferably, an isolated nucleic acid molecule of the invention thathybridizes under stringent conditions to the sequence of SEQ ID NO:1,SEQ ID NO:4 corresponds to a naturally-occurring nucleic acid molecule.As used herein, a “naturally-occurring” nucleic acid molecule refers toan RNA or DNA molecule having a nucleotide sequence that occurs innature (e.g., encodes a natural protein). In one embodiment, the nucleicacid encodes a natural human PGC-1.

In addition to naturally-occurring allelic variants of the PGC-1sequence that may exist in the population, the skilled artisan willfurther appreciate that changes can be introduced by mutation into thenucleotide sequence of SEQ ID NO:1, SEQ ID NO:4, thereby leading tochanges in the amino acid sequence of the encoded PGC-1 protein, withoutaltering the functional ability of the PGC-1 protein. For example,nucleotide substitutions leading to amino acid substitutions at“non-essential” amino acid residues can be made in the sequence of SEQID NO:1, SEQ ID NO:4. A “non-essential” amino acid residue is a residuethat can be altered from the wild-type sequence of PGC-1 (e.g., thesequence of SEQ ID NO:2, SEQ ID NO:5) without altering the activity ofPGC-1, whereas an “essential” amino acid residue is required for PGC-1activity. For example, amino acid residues involved in the interactionof PGC-1 to PPARγ are most likely essential residues of PGC-1. Otheramino acid residues, however, (e.g., those that are not conserved oronly semi-conserved between mouse and human) may not be essential foractivity and thus are likely to be amenable to alteration withoutaltering PGC-1 activity.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding PGC-1 proteins that contain changes in amino acidresidues that are not essential for PGC-1 activity. Such PGC-1 proteinsdiffer in amino acid sequence from SEQ ID NO:2, SEQ ID NO:5 yet retainat least one of the PGC-1 activities described herein. In oneembodiment, the isolated nucleic acid molecule comprises a nucleotidesequence encoding a protein, wherein the protein comprises an amino acidsequence at least about 60% homologous to the amino acid sequence of SEQID NO:2, SEQ ID NO:5 and is capable of modulating differentiation ofwhite adipocytes to brown adipocytes and/or thermogenesis of brownadipocytes. Preferably, the protein encoded by the nucleic acid moleculeis at least about 70% homologous, preferably at least about 80-85%homologous, still more preferably at least about 90%, and mostpreferably at least about 95% homologous to the amino acid sequence ofSEQ ID NO:2, SEQ ID NO:5.

“Sequence identity or homology”, as used herein, refers to the sequencesimilarity between two polypeptide molecules or between two nucleic acidmolecules. When a position in both of the two compared sequences isoccupied by the same base or amino acid monomer subunit, e.g., if aposition in each of two DNA molecules is occupied by adenine, then diemolecules are homologous or sequence identical at that position. Thepercent of homology or sequence identity between two sequences is afunction of the number of matching or homologous identical positionsshared by the two sequences divided by the number of positions comparedx 100. For example, if 6 of 10, of the positions in two sequences arethe same then the two sequences are 60% homologous or have 60% sequenceidentity. By way of example, the DNA sequences ATTGCC and TATGGC share50% homology or sequence identity. Generally, a comparison is made whentwo sequences are aligned to give maximum homology. Unless otherwisespecified “loop out regions”, e.g., those arising from, from deletionsor insertions in one of the sequences are counted as mismatches.

The comparison of sequences and determination of percent homologybetween two sequences can be accomplished using a mathematicalalgorithm. Preferably, the alignment can be performed using the ClustalMethod. Multiple alignment parameters include GAP Penalty=10, Gap LengthPenalty=10. For DNA alignments, the pairwise alignment parameters can beHtuple=2, Gap penalty=5, Window=4, and Diagonal saved=4. For proteinalignments, the pairwise alignment parameters can be Ktuple=1, Gappenalty=3, Window=5, and Diagonals Saved=5.

In a preferred embodiment, the percent identity between two amino acidsequences is determined using the Needleman and Wunsch (J. Mol. Biol.(48):444-453 (1970)) algorithm which has been incorporated into the GAPprogram in the GCG software package (available at http://www.gcg.com),using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.In yet another preferred embodiment, the percent identity between twonucleotide sequences is determined using the GAP program in the GCGsoftware package (available at http://www.gcg.com), using aNWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and alength weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percentidentity between two amino acid or nucleotide sequences is determinedusing the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)which has been incorporated into the ALIGN program (version 2.0)(available at http://vega.igh.cnrs.fr/bin/align-guess.cgi), using aPAM120 weight residue table, a gap length penalty of 12 and a gappenalty of 4.

An isolated nucleic acid molecule encoding a PGC-1 protein homologous tothe protein of SEQ ID NO:2, SEQ ID NO:5 can be created by introducingone or more nucleotide substitutions, additions or deletions into thenucleotide sequence of SEQ ID NO:1, SEQ ID NO:4 or a homologousnucleotide sequence such that one or more amino acid substitutions,additions or deletions are introduced into the encoded protein.Mutations can be introduced into SEQ ID NO:1, SEQ ID NO:4 or thehomologous nucleotide sequence by standard techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis. Preferably,conservative amino acid substitutions are made at one or more predictednon-essential amino acid residues. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art. Thesefamilies include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a predicted nonessentialamino acid residue in PGC-1 is preferably replaced with another aminoacid residue from the same side chain family. Alternatively, in anotherembodiment, mutations can be introduced randomly along all or part of aPGC-1 coding sequence, such as by saturation mutagenesis, and theresultant mutants can be screened for a PGC-1 activity described hereinto identify mutants that retain PGC-1 activity. Following mutagenesis ofSEQ ID NO:1, SEQ ID NO:4, the encoded protein can be expressedrecombinantly (e.g., as described in Example IV) and the activity of theprotein can be determined using, for example, assays described herein.

In addition to the nucleic acid molecules encoding PGC-1 proteinsdescribed above, another aspect of the invention pertains to isolatednucleic acid molecules which are antisense thereto. An “antisense”nucleic acid comprises a nucleotide sequence which is complementary to a“sense” nucleic acid encoding a protein, e.g., complementary to thecoding strand of a double-stranded cDNA molecule or complementary to anmRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bondto a sense nucleic acid. The antisense nucleic acid can be complementaryto an entire PGC-1 coding strand, or to only a portion thereof. In oneembodiment, an antisense nucleic acid molecule is antisense to a “codingregion” of the coding strand of a nucleotide sequence encoding PGC-1.The term “coding region” refers to the region of the nucleotide sequencecomprising codons which are translated into amino acid residues (e.g.,the entire coding region of SEQ ID NO:1 comprises nucleotides 92 to2482, the entire coding region of SEQ ID NO:4 comprises nucleotides 89to 2482). In another embodiment, the antisense nucleic acid molecule isantisense to a “noncoding region” of the coding strand of a nucleotidesequence encoding PGC-1. The term “noncoding region” refers to 5′ and 3′sequences which flank the coding region that are not translated intoamino acids (i.e., also referred to as 5′ and 3′ untranslated regions).

Given the coding strand sequences encoding PGC-1 disclosed herein (e.g.,SEQ ID NO:1, SEQ ID NO:4), antisense nucleic acids of the invention canbe designed according to the rules of Watson and Crick base pairing. Theantisense nucleic acid molecule can be complementary to the entirecoding region of PGC-1 mRNA, but more preferably is an oligonucleotidewhich is antisense to only a portion of the coding or noncoding regionof PGC-1 mRNA. For example, the antisense oligonucleotide can becomplementary to the region surrounding the translation start site ofPGC-1 mRNA. An antisense oligonucleotide can be, for example, about 5,10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisensenucleic acid of the invention can be constructed using chemicalsynthesis and enzymatic ligation reactions using procedures known in theart. For example, an antisense nucleic acid (e.g., an antisenseoligonucleotide) can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed between the antisense and sense nucleicacids, e.g., phosphorothioate derivatives and acridine substitutednucleotides can be used. Examples of modified nucleotides which can beused to generate the antisense nucleic acid include 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a PGC-1protein to thereby inhibit expression of the protein, e.g., byinhibiting transcription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. An example of a route of administration of anantisense nucleic acid molecule of the invention includes directinjection at a tissue site. Alternatively, an antisense nucleic acidmolecule can be modified to target selected cells and then administeredsystemically. For example, for systemic administration, an antisensemolecule can be modified such that it specifically binds to a receptoror an antigen expressed on a selected cell surface, e.g., by linking theantisense nucleic acid molecule to a peptide or an antibody which bindsto a cell surface receptor or antigen. The antisense nucleic acidmolecule can also be delivered to cells using the vectors describedherein. To achieve sufficient intracellular concentrations of theantisense molecules, vector constructs in which the antisense nucleicacid molecule is placed under the control of a strong pol II or pol IIIpromoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

In still another embodiment, an antisense nucleic acid of the inventionis a ribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity which are capable of cleaving a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. Thus,ribozymes (e.g., hammerhead ribozymes (described in Haseloff and Gerlach(1988) Nature 334:585-591)) can be used to catalytically cleave PGC-1mRNA transcripts to thereby inhibit translation of PGC-1 mRNA. Aribozyme having specificity for a PGC-1-encoding nucleic acid can bedesigned based upon the nucleotide sequence of a PGC-1 cDNA disclosedherein (e.g., SEQ ID NO:1, SEQ ID NO:4). For example, a derivative of aTetrahymena L-19 IVS RNA can be constructed in which the nucleotidesequence of the active site is complementary to the nucleotide sequenceto be cleaved in a PGC-1-encoding mRNA. See, e.g., Cech et al. U.S. Pat.No. 4,987,071 and Cech et al. U.S. Pat. No. 5,116,742. Alternatively,PGC-1 mRNA can be used to select a catalytic RNA having a specificribonuclease activity from a pool of RNA molecules. See, e.g., Bartel,D. and Szostak, J. W. (1993) Science 261:1411-1418.

Alternatively, PGC-1 gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of the PGC-l(e.g., the PGC-1 promoter and/or enhancers) to form triple helicalstructures that prevent transcription of the PGC-1 gene in target cells.See generally, Helene, C. (1991) Anticancer Drug Des. 6(6):569-84;Helene, C. et al. (1992) Ann. N. Y Acad Sci. 660:27-36; and Maher, L. J.(1992) Bioassays 14(12):807-15.

II. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding PGC-1 (or aportion thereof). As used herein, the term “vector” refers to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments canbe ligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “expression vectors”. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to includes promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel; Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those which direct constitutive expression of anucleotide sequence in many types of host cell and those which directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, etc. The expression vectors ofthe invention can be introduced into host cells to thereby produceproteins or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein (e.g., PGC-1 proteins, mutant forms ofPGC-1, fusion proteins, etc.).

The recombinant expression vectors of the invention can be designed forexpression of PGC-1 in prokaryotic or eukaryotic cells. For example,PGC-1 can be expressed in bacterial cells such as E. coli, insect cells(using baculovirus expression vectors) yeast cells or mammalian cells.Suitable host cells are discussed further in Goeddel, Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990). Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67:3140), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein. In oneembodiment, the coding sequence of the PGC-1 is cloned into a pGEXexpression vector to create a vector encoding a fusion proteincomprising, from the N-terminus to the C-terminus, GST-thrombin cleavagesite-PGC-1. The fusion protein can be purified by affinitychromatography using glutathione-agarose resin. Recombinant PGC-1unfused to GST can be recovered by cleavage of the fusion protein withthrombin.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d (Studieret al., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 60-89). Target gene expression from thepTrc vector relies en host RNA polymerase transcription from a hybridtrp-lac fusion promoter. Target gene expression from the pET 11d vectorrelies on transcription from a T7 gn10-lac fusion promoter mediated by acoexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21 (DE3) or HMS174(DE3) from a resident λprophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV 5 promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al. (1992) Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the PGC-1 expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerivisae includepYepSecl (Baldari, et al., (1987) EMBO J. 6:229-234), pMFa (Kurjan andHerskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene54:113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif.

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, B. (1987) Nature329:840) and pMT2PC (Kaufman et al. (1987) EMBO J 6:187-195). When usedin mammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol. 43:235-275), in particular promoters of Tcell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) andimmunoglobulins (Banedji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) PNAS 86:5473-5477),pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916),and mammary gland-specific promoters (e.g., milk whey promoter; U.S.Pat. No. 4,873,316 and European Application Publication No. 264,166).Developmentally-regulated promoters are also encompassed, for examplethe murine hox promoters (Kessel and Gruss (1990) Science 249:374-379)and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev.3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to PGC-1 mRNA. Regulatory sequences operativelylinked to a nucleic acid cloned in the antisense orientation can bechosen which direct the continuous expression of the antisense RNAmolecule in a variety of cell types, for instance viral promoters and/orenhancers, or regulatory sequences can be chosen which directconstitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes see Weintraub, H. etal., Antisense RNA as a molecular tool for genetic analysis,Reviews—Trends in Genetics, Vol. 1(1) 1986.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein, it is understood that such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example,PGC-1 protein can be expressed in bacterial cells such as E. coli,insect cells, yeast or mammalian cells (such as Chinese hamster ovarycells (CHO) or COS cells). Other suitable host cells are known to thoseskilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (MolecularCloning: A Laboratory Manual 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Nucleic acid encodinga selectable marker can be introduced into a host cell on the samevector as that encoding PGC-1 or can be introduced on a separate vector.Cells stably transfected with the introduced nucleic acid can beidentified by drug selection (e.g., cells that have incorporated theselectable marker gene will survive, while the other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) PGC-1 protein.Accordingly, the invention further provides methods for producing PGC-1protein using the host cells of the invention. In one embodiment, themethod comprises culturing the host cell of invention (into which arecombinant expression vector encoding PGC-1 has been introduced) in asuitable medium until PGC-1 is produced. In another embodiment, themethod further comprises isolating PGC-1 from the medium or the hostcell.

The host cells of the invention can also be used to produce nonhumantransgenic animals. The nonhuman transgenic animals can be used inscreening assays designed to identify agents or compounds, e.g., drugs,pharmaceuticals, etc., which are capable of ameliorating detrimentalsymptoms of selected disorders such as weight disorders or disordersassociated with insufficient insulin activity. For example, in oneembodiment, a host cell of the invention is a fertilized oocyte or anembryonic stem cell into which PGC-1-coding sequences have beenintroduced. Such host cells can then be used to create non-humantransgenic animals in which exogenous PGC-1 sequences have beenintroduced into their genome or homologous recombinant animals in whichendogenous PGC-1 sequences have been altered. Such animals are usefulfor studying the function and/or activity of PGC-1 and for identifyingand/or evaluating modulators of PGC-1 activity. As used herein, a“transgenic animal” is a nonhuman animal, preferably a mammal, morepreferably a rodent such as a rat or mouse, in which one or more of thecells of the animal includes a transgene. Other examples of transgenicanimals include nonhuman primates, sheep, dogs, cows, goats, chickens,amphibians, etc. A transgene is exogenous DNA which is integrated intothe genome of a cell from which a transgenic animal develops and whichremains in the genome of the mature animal, thereby directing theexpression of an encoded gene product in one or more cell types ortissues of the transgenic animal. As used herein, a “homologousrecombinant animal” is a nonhuman animal, preferably a mammal, morepreferably a mouse, in which an endogenous PGC-1 gene has been alteredby homologous recombination between the endogenous gene and an exogenousDNA molecule introduced into a cell of the animal, e.g., an embryoniccell of the animal, prior to development of the animal.

A transgenic animal of the invention can be created by introducingPGC-1-encoding nucleic acid into the male pronuclei of a fertilizedoocyte, e.g., by microinjection, retroviral infection, and allowing theoocyte to develop in a pseudopregnant female foster animal. The humanPGC-1 cDNA sequence can be introduced as a transgene into the genome ofa nonhuman animal. Alternatively, a nonhuman homologue of the humanPGC-1 gene (SEQ ID NO:4), such as a mouse PGC-1 gene (SEQ ID NO:1), canused as a transgene. Intronic sequences and polyadenylation signals canalso be included in the transgene to increase the efficiency ofexpression of the transgene. A tissue-specific regulatory sequence(s)can be operably linked to the PGC-1 transgene to direct expression ofPGC-1 protein to particular cells. Methods for generating transgenicanimals via embryo manipulation and microinjection, particularly animalssuch as mice, have become conventional in the art and are described, forexample, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder etal., U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B.,Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1986). Similar methods are used for productionof other transgenic animals. A transgenic founder animal can beidentified based upon the presence of the PGC-1 transgene in its genomeand/or expression of PGC-1 mRNA in tissues or cells of the animals. Atransgenic founder animal can then be used to breed additional animalscarrying the transgene. Moreover, transgenic animals carrying atransgene encoding PGC-1 can further be bred to other transgenic animalscarrying other transgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of a PGC-1 gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the PGC-1 gene. The PGC-1 gene can be a human gene(e.g., from a human genomic clone isolated from a human genomic libraryscreened with the cDNA of SEQ ID NO:1), but more preferably, is anonhuman homologue of a human PGC-1 gene. For example, a mouse PGC-1gene can be used to construct a homologous recombination vector suitablefor altering an endogenous PGC-1 gene in the mouse genome. In apreferred embodiment, the vector is designed such that, upon homologousrecombination, the endogenous PGC-1 gene is functionally disrupted(i.e., no longer encodes a functional protein; also referred to as a“knock out” vector). Alternatively, the vector can be designed suchthat, upon homologous recombination, the endogenous PGC-1 gene ismutated or otherwise altered but still encodes functional protein (e.g.,the upstream regulatory region can be altered to thereby alter theexpression of the endogenous PGC-1 protein). In the homologousrecombination vector, the altered portion of the PGC-1 gene is flankedat its 5′ and 3′ ends by additional nucleic acid of the PGC-1 gene toallow for homologous recombination to occur between the exogenous PGC-1gene carried by the vector and an endogenous PGC-1 gene in an embryonicstem cell. The additional flanking PGC-1 nucleic acid is of sufficientlength for successful homologous recombination with the endogenous gene.Typically, several kilobases of flanking DNA (both at the 5′ and 3′ends) are included in the vector (see e.g., Thomas, K. R. and Capecchi,M. R. (1987) Cell 51:503 for a description of homologous recombinationvectors). The vector is introduced into an embryonic stem cell line(e.g., by electroporation) and cells in which the introduced PGC-1 genehas homologously recombined with the endogenous PGC-1 gene are selected(see e.g., Li, E. et al. (1992) Cell 69:915). The selected cells arethen injected into a blastocyst of an animal (e.g., a mouse) to formaggregation chimeras (see e.g., Bradley, A. in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,Oxford, 1987) pp. 113-152). A chimeric embryo can then be implanted intoa suitable pseudopregnant female foster animal and the embryo brought toterm. Progeny harboring the homologously recombined DNA in their germcells can be used to breed animals in which all cells of the animalcontain the homologously recombined DNA by germline transmission of thetransgene. Methods for constructing homologous recombination vectors andhomologous recombinant animals are described further in Bradley, A.(1991) Current Opinion in Biotechnology 2:823-829 and in PCTInternational Publication Nos.: WO 90/11354 by Le Mouellec et al.; WO91/01140 by Smithies et al.; WO 92/0968 by Zijlstra et al.; and WO93/04169 by Bems et al.

In another embodiment, transgenic nonhuman animals can be produced whichcontain selected systems which allow for regulated expression of thetransgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad.Sci. USA 89:6232-6236. Another example of a recombinase system is theFLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al.(1991) Science 251:1351-1355. If a cre/loxP recombinase system is usedto regulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein are required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

Clones of the nonhuman transgenic animals described herein can also beproduced according to the methods described in Wilmut, l. et al. (1997)Nature 385:810-813 and PCT International Publication Nos. WO 97/07668and WO 97/07669. In brief, a cell, e.g., a somatic cell, from thetransgenic animal can be isolated and induced to exit the growth cycleand enter G₀ phase. The quiescent cell can then be fused, e.g., throughthe use of electrical pulses, to an enucleated oocyte from an animal ofthe same species from which the quiescent cell is isolated. Thereconstructed oocyte is then cultured such that it develops to morula orblastocyst and then transferred to pseudopregnant female foster animal.The offspring borne of this female foster animal will be a clone of theanimal from which the cell, e.g., the somatic cell, is isolated.

III. Isolated PGC-1 Proteins and Anti-PGC-1 Antibodies

Another aspect of the invention pertains to isolated PGC-1 proteins, andbiologically active portions thereof, as well as peptide fragmentssuitable for use as immunogens to raise anti-PGC-1 antibodies. An“isolated” or “purified” protein or biologically active portion thereofis substantially free of cellular material when produced by recombinantDNA techniques, or chemical precursors or other chemicals whenchemically synthesized. The language “substantially free of cellularmaterial” includes preparations of PGC-1 protein in which the protein isseparated from cellular components of the cells in which it is naturallyor recombinantly produced. In one embodiment, the language“substantially free of cellular material” includes preparations of PGC-1protein having less than about 30% (by dry weight) of non-PGC-1 protein(also referred to herein as a “contaminating protein”), more preferablyless than about 20% of non-PGC-1 protein, still more preferably lessthan about 10% of non-PGC-1 protein, and most preferably less than about5% non-PGC-1 protein. When the PGC-1 protein or biologically activeportion thereof is recombinantly produced, it is also preferablysubstantially free of culture medium, i.e., culture medium representsless than about 20%, more preferably less than about 10%, and mostpreferably less than about 5% of the volume of the protein preparation.The language “substantially free of chemical precursors or otherchemicals” includes preparations of PGC-1 protein in which the proteinis separated from chemical precursors or other chemicals which areinvolved in the synthesis of the protein. In one embodiment, thelanguage “substantially free of chemical precursors or other chemicals”includes preparations of PGC-1 protein having less than about 30% (bydry weight) of chemical precursors or non-PGC-1 chemicals, morepreferably less than about 20% chemical precursors or non-PGC-1chemicals, still more preferably less than about 10% chemical precursorsor non-PGC-1 chemicals, and most preferably less than about 5% chemicalprecursors or non-PGC-1 chemicals. In preferred embodiments, isolatedproteins or biologically active portions thereof lack contaminatingproteins from the same animal from which the PGC-1 protein is derived.Typically, such proteins are produced by recombinant expression of, forexample, a human PGC-1 protein in a nonhuman cell.

An isolated PGC-1 protein or a portion thereof of the invention has oneor more of the following biological activities: 1) it can interact with(e.g., bind to) PPARγ; 2) it can modulate PPARγ activity; 3) it canmodulate UCP expression; 4) it can modulate thermogenesis in adipocytes,e.g., thermogenesis in brown adipocytes, or muscle; 5) it can modulateoxygen consumption in adipocytes or muscle; 6) it can modulateadipogenesis, e.g., differentiation of white adipocytes into brownadipocytes; 7) it can modulate insulin sensitivity of cells, e.g.,insulin sensitivity of muscle cells, liver cells, adipocytes; 8) it caninteract with (e.g., bind to) nuclear hormone receptors, e.g., thethyroid hormone receptor, the estrogen receptor, the retinoic acidreceptor; 9) it can modulate the activity of nuclear hormone receptors;and 10) it can interact with (e.g., bind to) the transcription factorC/EBPα. In a preferred embodiment, the PGC-1 protein can modulatedifferentiation of white adipocytes to brown adipocytes and/orthermogenesis in brown adipocytes or muscle cells.

In preferred embodiments, the protein or portion thereof comprises anamino acid sequence which is sufficiently homologous to an amino acidsequence of SEQ ID NO:2, SEQ ID NO:5 such that the protein or portionthereof maintains the ability to modulate differentiation of adipocytesand/or thermogenesis in brown adipocytes. The portion of the protein ispreferably a biologically active portion as described herein. In anotherpreferred embodiment, the PGC-1 protein (i.e., amino acid residues 1-797and amino acid residues1-798) has an amino acid sequence shown in SEQ IDNO:2, SEQ ID NO:5, respectively, or an amino acid sequence which is atleast about 50%, preferably at least about 60%, more preferably at leastabout 70%, yet more preferably at least about 80%, still more preferablyat least about 90%, and most preferably at least about 95% or morehomologous to the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:5.In yet another preferred embodiment, the PGC-1 protein has an amino acidsequence which is encoded by a nucleotide sequence which hybridizes,e.g., hybridizes under stringent conditions, to the nucleotide sequenceof SEQ ID NO:1, SEQ ID NO:4 or a nucleotide sequence which is at leastabout 50%, preferably at least about 60%, more preferably at least about70%, yet more preferably at least about 80%, still more preferably atleast about 90%, and most preferably at least about 95% or morehomologous to the nucleotide sequence shown in SEQ ID NO: I, SEQ IDNO:4. The preferred PGC-1 proteins of the present invention alsopreferably possess at least one of the PGC-1 biological activitiesdescribed herein. For example, a preferred PGC-1 protein of the presentinvention includes an amino acid sequence encoded by a nucleotidesequence which hybridizes, e.g., hybridizes under stringent conditions,to the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:4 and which canmodulate differentiation of white adipocytes to brown adipocytes and/orthermogenesis of brown adipocytes.

In other embodiments, the PGC-1 protein is substantially homologous tothe amino acid sequence of SEQ ID NO:2, SEQ ID NO:5 and retains thefunctional activity of the protein of SEQ ID NO:2, SEQ ID NO:5 yetdiffers in amino acid sequence due to natural allelic variation ormutagenesis, as described in detail in subsection I above. Accordingly,in another embodiment, the PGC-1 protein is a protein which comprises anamino acid sequence which is at least about 50%, preferably at leastabout 60%, more preferably at least about 70%, yet more preferably atleast about 80%, still more preferably at least about 90%, and mostpreferably at least about 95% or more homologous to the amino acidsequence of SEQ ID NO:2, SEQ ID NO:5.

Biologically active portions of the PGC-1 protein include peptidescomprising amino acid sequences derived from the amino acid sequence ofthe PGC-1 protein, e.g., the amino acid sequence shown in SEQ ID NO:2,SEQ ID NO:5 or the amino acid sequence of a protein homologous to thePGC-1 protein, which include less amino acids than the full length PGC-1protein or the full length protein which is homologous to the PGC-1protein, and exhibit at least one activity of the PGC-1 protein.Typically, biologically active portions (peptides, e.g., peptides whichare, for example, 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 ormore amino acids in length) comprise a domain or motif, e.g., a tyrosinephosphorylation site, a cAMP phosphorylation site, a serine-arginine(SR) rich domain, and/or an RNA binding motif, with at least oneactivity of the PGC-1 protein. In a preferred embodiment, thebiologically active portion of the protein which includes one or morethe domains/motifs described herein can modulate differentiation ofadipocytes and/or thermogenesis in brown adipocytes. Moreover, otherbiologically active portions, in which other regions of the protein aredeleted, can be prepared by recombinant techniques and evaluated for oneor more of the activities described herein. Preferably, the biologicallyactive portions of the PGC-1 protein include one or more selecteddomains/motifs or portions thereof having biological activity.

PGC-1 proteins are preferably produced by recombinant DNA techniques.For example, a nucleic acid molecule encoding the protein is cloned intoan expression vector (as described above), the expression vector isintroduced into a host cell (as described above) and the PGC-1 proteinis expressed in the host cell. The PGC-1 protein can then be isolatedfrom the cells by an appropriate purification scheme using standardprotein purification techniques. Alternative to recombinant expression,a PGC-1 protein, polypeptide, or peptide can be synthesized chemicallyusing standard peptide synthesis techniques. Moreover, native PGC-1protein can be isolated from cells (e.g., brown adipocytes), for exampleusing an anti-PGC-1 antibody (described further below).

The invention also provides PGC-1 chimeric or fusion proteins. As usedherein, a PGC-1 “chimeric protein” or “fusion protein” comprises a PGC-1polypeptide operatively linked to a non-PGC-1 polypeptide. A“PGC-1polypeptide” refers to a polypeptide having an amino acid sequencecorresponding to PGC-1, whereas a “non-PGC-1 polypeptide” refers to apolypeptide having an amino acid sequence corresponding to a proteinwhich is not substantially homologous to the PGC-1 protein, e.g., aprotein which is different from the PGC-1 protein and which is derivedfrom the same or a different organism. Within the fusion protein, theterm “operatively linked” is intended to indicate that the PGC-1polypeptide and the non-PGC-1 polypeptide are fused in-frame to eachother. The non-PGC-1 polypeptide can be fused to the N-terminus orC-terminus of the PGC-1 polypeptide. For example, in one embodiment thefusion protein is a GST-PGC-1 fusion protein in which the PGC-1sequences are fused to the C-terminus of the GST sequences (see ExampleIV). Such fusion proteins can facilitate the purification of recombinantPGC-1. In another embodiment, the fusion protein is a PGC-1 proteincontaining a heterologous signal sequence at its N-terminus. In certainhost cells (e.g., mammalian host cells), expression and/or secretion ofPGC-1 can be increased through use of a heterologous signal sequence.

Preferably, a PGC-1 chimeric or fusion protein of the invention isproduced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, forexample by employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and enzymatic ligation. Inanother embodiment, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed and reamplified to generatea chimeric gene sequence (see, for example, Current Protocols inMolecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). APGC-1-encoding nucleic acid can be cloned into such an expression vectorsuch that the fusion moiety is linked in-frame to the PGC-1 protein.

The present invention also pertains to homologues of the PGC-1 proteinswhich function as either a PGC-1 agonist (mimetic) or a PGC-1antagonist. In a preferred embodiment, the PGC-1 agonists andantagonists stimulate or inhibit, respectively, a subset of thebiological activities of the naturally occurring form of the PGC-1protein. Thus, specific biological effects can be elicited by treatmentwith a homologue of limited function. In one embodiment, treatment of asubject with a homologue having a subset of the biological activities ofthe naturally occurring form of the protein has fewer side effects in asubject relative to treatment with the naturally occurring form of thePGC-1 protein.

Homologues of the PGC-1 protein can be generated by mutagenesis, e.g.,discrete point mutation or truncation of the PGC-1 protein. As usedherein, the term “homologue” refers to a variant form of the PGC-1protein which acts as an agonist or antagonist of the activity of thePGC-1 protein. An agonist of the PGC-1 protein can retain substantiallythe same, or a subset, of the biological activities of the PGC-1protein. An antagonist of the PGC-1 protein can inhibit one or more ofthe activities of the naturally occurring form of the PGC-1 protein, by,for example, competitively binding to a downstream or upstream member ofthe PGC-1 cascade which includes the PGC-1 protein. Thus, the mammalianPGC-1 protein and homologues thereof of the present invention can be,for example, either positive or negative regulators of adipocytedifferentiation and/or thermogenesis in brown adipocytes.

In an alternative embodiment, homologues of the PGC-1 protein can beidentified by screening combinatorial libraries of mutants, e.g.,truncation mutants, of the PGC-1 protein for PGC-1 protein agonist orantagonist activity. In one embodiment, a variegated library of PGC-1variants is generated by combinatorial mutagenesis at the nucleic acidlevel and is encoded by a variegated gene library. A variegated libraryof PGC-1 variants can be produced by, for example, enzymaticallyligating a mixture of synthetic oligonucleotides into gene sequencessuch that a degenerate set of potential PGC-1 sequences is expressibleas individual polypeptides, or alternatively, as a set of larger fusionproteins (e.g., for phage display) containing the set of PGC-1 sequencestherein. There are a variety of methods which can be used to producelibraries of potential PGC-1 homologues from a degenerateoligonucleotide sequence. Chemical synthesis of a degenerate genesequence can be performed in an automatic DNA synthesizer, and thesynthetic gene then ligated into an appropriate expression vector. Useof a degenerate set of genes allows for the provision, in one mixture,of all of the sequences encoding the desired set of potential PGC-1sequences. Methods for synthesizing degenerate oligonucleotides areknown in the art (see, e.g., Narang, S. A. (1983) Tetrahedron 39:3;Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984)Science 198:1056; Ike et al. (1983) Nucleic Acid Res 1 1:477.

In addition, libraries of fragments of the PGC-1 protein coding can beused to generate a variegated population of PGC-1 fragments forscreening and subsequent selection of homologues of a PGC-1 protein. Inone embodiment, a library of coding sequence fragments can be generatedby treating a double stranded PCR fragment of a PGC-1 coding sequencewith a nuclease under conditions wherein nicking occurs only about onceper molecule, denaturing the double stranded DNA, renaturing the DNA toform double stranded DNA which can include sense/antisense pairs fromdifferent nicked products, removing single stranded portions fromreformed duplexes by treatment with S1 nuclease, and ligating theresulting fragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal, C-terminaland internal fragments of various sizes of the PGC-1 protein.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of PGC-1 homologues. The mostwidely used techniques, which are amenable to high through-put analysis,for screening large gene libraries typically include cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a newtechnique which enhances the frequency of functional mutants in thelibraries, can be used in combination with the screening assays toidentify PGC-1 homologues (Arkin and Youvan (1992) Proc. Natl. Acad.Sci. USA 89:7811-7815; Delagrave et al. (1993) Protein Eng.6(3):327-331).

An isolated PGC-1 protein, or a portion or fragment thereof, can be usedas an immunogen to generate antibodies that bind PGC-1 using standardtechniques for polyclonal and monoclonal antibody preparation. Thefull-length PGC-1 protein can be used or, alternatively, the inventionprovides antigenic peptide fragments of PGC-1 for use as immunogens. Theantigenic peptide of PGC-1 comprises at least 8 amino acid residues ofthe amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:5 or ahomologous amino acid sequence as described herein and encompasses anepitope of PGC-1 such that an antibody raised against the peptide formsa specific immune complex with PGC-1. Preferably, the antigenic peptidecomprises at least 10 amino acid residues, more preferably at least 15amino acid residues, even more preferably at least 20 amino acidresidues, and most preferably at least 30 amino acid residues. Preferredepitopes encompassed by the antigenic peptide are regions of PGC-1 thatare located on the surface of the protein, e.g., hydrophilic regions.

A PGC-1 immunogen typically is used to prepare antibodies by immunizinga suitable subject, (e.g., rabbit, goat, mouse or other mammal) with theimmunogen. An appropriate immunogenic preparation can contain, forexample, recombinantly expressed PGC-1 protein or a chemicallysynthesized PGC-1 peptide. The preparation can further include anadjuvant, such as Freund's complete or incomplete adjuvant, or similarimmunostimulatory agent. Immunization of a suitable subject with animmunogenic PGC-1 preparation induces a polyclonal anti-PGC-1 antibodyresponse.

Accordingly, another aspect of the invention pertains to anti-PGC-1antibodies. The term “antibody” as used herein refers to immunoglobulinmolecules and immunologically active portions of immunoglobulinmolecules, i.e., molecules that contain an antigen binding site whichspecifically binds (immunoreacts with) an antigen, such as PGC-1.Examples of immunologically active portions of immunoglobulin moleculesinclude F(ab) and F(ab′)₂ fragments which can be generated by treatingthe antibody with an enzyme such as pepsin. The invention providespolyclonal and monoclonal antibodies that bind PGC-1. The term“monoclonal antibody” or “monoclonal antibody composition”, as usedherein, refers to a population of antibody molecules that contain onlyone species of an antigen binding site capable of immunoreacting with aparticular epitope of PGC-1. A monoclonal antibody composition thustypically displays a single binding affinity for a particular PGC-1protein with which it immunoreacts.

Polyclonal anti-PGC-1 antibodies can be prepared as described above byimmunizing a suitable subject with a PGC-1 immunogen. The anti-PGC-1antibody titer in the immunized subject can be monitored over time bystandard techniques, such as with an enzyme linked immunosorbent assay(ELISA) using immobilized PGC-1. If desired, the antibody moleculesdirected against PGC-1 can be isolated from the mammal (e.g., from theblood) and further purified by well known techniques, such as protein Achromatography to obtain the IgG fraction. At an appropriate time afterimmunization, e.g., when the anti-PGC-1 antibody titers are highest,antibody-producing cells can be obtained from the subject and used toprepare monoclonal antibodies by standard techniques, such as thehybridoma technique originally described by Kohler and Milstein (1975)Nature 256:495-497) (see also, Brown et al. (1981) J. Immunol.127:539-46; Brown et al. (1980) J. Biol. Chem 255:4980-83; Yeh et al.(1976) PNAS 76:2927-31; and Yeh et al. (1982) Int. J Cancer 29:269-75),the more recent human B cell hybridoma technique (Kozbor et al. (1983)Immunol Today 4:72), the EBV-hybridoma technique (Cole et al. (1985),Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96)or trioma techniques. The technology for producing monoclonal antibodyhybridomas is well known (see generally R. H. Kenneth, in MonoclonalAntibodies: A New Dimension In Biological Analyses, Plenum PublishingCorp., New York, N.Y. (1980); E. A. Lemer (1981) Yale J. Biol. Med,54:387402; M. L. Gefter et al. (1977) Somatic Cell Genet. 3:231-36).Briefly, an immortal cell line (typically a myeloma) is fused tolymphocytes (typically splenocytes) from a mammal immunized with a PGC-1immunogen as described above, and the culture supernatants of theresulting hybridoma cells are screened to identify a hybridoma producinga monoclonal antibody that binds PGC-1.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating ananti-PGC-I monoclonal antibody (see, e.g., G. Galfre et al. (1977)Nature 266:55052; Gefter et al. Somatic Cell Genet., cited supra; Lemer,Yale J. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies, citedsupra). Moreover, the ordinarily skilled worker will appreciate thatthere are many variations of such methods which also would be useful.Typically, the immortal cell line (e.g., a myeloma cell line) is derivedfrom the same mammalian species as the lymphocytes. For example, murinehybridomas can be made by fusing lymphocytes from a mouse immunized withan immunogenic preparation of the present invention with an immortalizedmouse cell line. Preferred immortal cell lines are mouse myeloma celllines that are sensitive to culture medium containing hypoxanthine,aminopterin and thymidine (“HAT medium”). Any of a number of myelomacell lines can be used as a fusion partner according to standardtechniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14myeloma lines. These myeloma lines are available from ATCC. Typically,HAT-sensitive mouse myeloma cells are fused to mouse splenocytes usingpolyethylene glycol (“PEG”). Hybridoma cells resulting from the fusionare then selected using HAT medium, which kills unfused andunproductively fused myeloma cells (unflused splenocytes die afterseveral days because they are not transformed). Hybridoma cellsproducing a monoclonal antibody of the invention are detected byscreening the hybridoma culture supernatants for antibodies that bindPGC-1, e.g., using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal anti-PGC-1 antibody can be identified and isolated byscreening a recombinant combinatorial immunoglobulin library (e.g., anantibody phage display library) with PGC-1 to thereby isolateimmunoglobulin library members that bind PGC-1. Kits for generating andscreening phage display libraries are commercially available (e.g., thePharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; andthe Stratagene SurJZAP™ Phage Display Kit, Catalog No. 240612).Additionally, examples of methods and reagents particularly amenable foruse in generating and screening antibody display library can be foundin, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCTInternational Publication No. WO 92/18619; Dower et al. PCTInternational Publication No. WO 91/17271; Winter et al. PCTInternational Publication WO 92/20791; Markland et al. PCT InternationalPublication No. WO 92/15679; Breitling et al. PCT InternationalPublication WO 93/01288; McCafferty et al. PCT International PublicationNo. WO 92/01047; Garrard et al. PCT International Publication No. WO92/09690; Ladner et al. PCT International Publication No. WO 90/02809;Fuchs et al. (1991) Bio/Technology 9:1369-1372; Hay-et al. (1992) Hum.Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;Griffiths et al. (1993) EMBO J J. 12:725-734; Hawkins et al. (1992) J.Mol. Biol. 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gramet al. (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrard et al.(1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) NucleicAcids Res. 19:4133-4137; Barbas et al. (1991) Proc. Natl. Acad. Sci. USA88:7978-7982; and McCafferty et al. Nature (1990)

Additionally, recombinant anti-PGC-1 antibodies, such as chimeric andhumanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in Robinson et al.International Application No. PCT/US86/02269; Akira, et al. EuropeanPatent Application 184,187; Taniguchi, M., European Patent Application171,496; Morrison et al. European Patent Application 173,494; Neubergeret al. PCT International Publication No. WO 86/01533; Cabilly et al.U.S. Pat. No. 4,816,567; Cabilly et al. European Patent Application125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987)Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol.139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218;Nishimura et al. (1987) Cancer Res. 47:999-1005; Wood et al. (1985)Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et al.(1986) BioTechniques 4:214; Winter U.S. Pat. No. 5,225,539; Jones et al.(1986) Nature 321:552-525; Verhoeyen et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060.

An anti-PGC-1 antibody (e.g., monoclonal antibody) can be used toisolate PGC-1 by standard techniques, such as affinity chromatography orimmunoprecipitation. An anti-PGC-1 antibody can facilitate thepurification of natural PGC-1 from cells and of recombinantly producedPGC-1 expressed in host cells. Moreover, an anti-PGC-1 antibody can beused to detect PGC-1 protein (e.g., in a cellular lysate or cellsupernatant) in order to evaluate the abundance and pattern ofexpression of the PGC-1 protein. Anti-PGC-1 antibodies can be useddiagnostically to monitor protein levels in tissue as part of a clinicaltesting procedure, e.g., to, for example, determine the efficacy of agiven treatment regimen. Detection can be facilitated by coupling (i.e.,physically linking) the antibody to a detectable substance. Examples ofdetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,and radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

IV. Pharmaceutical Compositions

The PGC-1 nucleic acid molecules, PGC-1 proteins, PGC-1 modulators, andanti-PGC-1 antibodies (also referred to herein as “active compounds”) ofthe invention can be incorporated into pharmaceutical compositionssuitable for administration to a subject, e.g., a human. Suchcompositions typically comprise the nucleic acid molecule, protein,modulator, or antibody and a pharmaceutically acceptable carrier. Asused herein the language “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration. Theuse of such media and agents for pharmaceutically active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active compound, such media can be used in thecompositions of the invention. Supplementary active compounds can alsobe incorporated into the compositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediamirietetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a. PGC-1 protein or anti-PGC-1 antibody) in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle which contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art.

The materials can also be obtained commercially from Alza Corporationand Nova Pharmaceuticals, Inc. Liposomal suspensions (includingliposomes targeted to infected cells with monoclonal antibodies to viralantigens) can also be used as pharmaceutically acceptable carriers.These can be prepared according to methods known to those skilled in theart, for example, as described in U.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) PNAS 91:3054-3057). Thepharmaceutical preparation of the gene therapy vector can include thegene therapy vector in an acceptable diluent, or can comprise a slowrelease matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g. retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

V. Uses and Methods of the Invention

The nucleic acid molecules, polypeptides, polypeptide homologues,modulators, and antibodies described herein can be used in one or moreof the following methods: 1) drug screening assays; 2) diagnosticassays; and 3) methods of treatment. A PGC-1 protein of the inventionhas one or more of the activities described herein and can thus be usedto, for example, modulate adipocyte differentiation, thermogenesis inbrown adipocytes, and insulin sensitivity in various cells, e.g., musclecells, liver cells, and adipocytes. The isolated nucleic acid moleculesof the invention can be used to express PGC-1 protein (e.g., via arecombinant expression vector in a host cell in gene therapyapplications), to detect PGC-1 mRNA (e.g., in a biological sample) or agenetic lesion in a PGC-1 gene, and to modulate PGC-1 activity, asdescribed further below. In addition, the PGC-1 proteins can be used toscreen drugs or compounds which modulate PGC-1 protein activity as wellas to treat disorders characterized by insufficient production of PGC-1protein or production of PGC-1 protein forms which have decreasedactivity compared to wild type PGC-1. Moreover, the anti-PGC-1antibodies of the invention can be used to detect and isolate PGC-1protein and modulate PGC-1 protein activity.

a. Drug Screening Assays:

The invention provides methods for identifying compounds or agents whichcan be used to treat disorders characterized by (or associated with)aberrant or abnormal PGC-1 nucleic acid expression and/or PGC-1polypeptide activity. These methods are also referred to herein as drugscreening assays and typically include the step of screening acandidate/test compound or agent for the ability to interact with (e.g.,bind to) a PGC-1 protein, to modulate the interaction of a PGC-1 proteinand a target molecule, and/or to modulate PGC-1 nucleic acid expressionand/or PGC-1 protein activity. Candidate/test compounds or agents whichhave one or more of these abilities can be used as drugs to treatdisorders characterized by aberrant or abnormal PGC-1 nucleic acidexpression and/or PGC-1 protein activity. Candidate/test compoundsinclude, for example, 1) peptides such as soluble peptides, includingIg-tailed fusion peptides and members of random peptide libraries (see,e.g., Lam, K. S. et al. (1991) Nature 354:82-84; Houghten, R. et al.(1991) Nature 354:84-86) and combinatorial chemistry-derived molecularlibraries made of D-and/or L-configuration amino acids; 2)phosphopeptides (e.g., members of random and partially degenerate,directed phosphopeptide libraries, see, e.g., Songyang, Z. et al. (1993)Cell 72:767-778); 3) antibodies (e.g., polyclonal, monoclonal,humanized, anti-idiotypic, chimeric, and single chain antibodies as wellas Fab, F(ab′)₂, Fab expression library fragments, and epitope-bindingfragments of antibodies); and 4) small organic and inorganic molecules(e.g., molecules obtained from combinatorial and natural productlibraries).

In one embodiment, the invention provides assays for screeningcandidate/test compounds which interact with (e.g., bind to) PGC-1protein. Typically, the assays are cell-free assays which include thesteps of combining a PGC-1 protein or a biologically active portionthereof, and a candidate/test compound, e.g., under conditions whichallow for interaction of (e.g., binding of) the candidate/test compoundto the PGC-1 protein or portion thereof to form a complex, and detectingthe formation of a complex, in which the ability of the candidatecompound to interact with (e.g., bind to) the PGC-1 polypeptide orfragment thereof is indicated by the presence of the candidate compoundin the complex. Formation of complexes between the PGC-1 protein and thecandidate compound can be quantitated, for example, using standardimmunoassays.

In another embodiment, the invention provides screening assays toidentify candidate/test compounds which modulate (e.g., stimulate orinhibit) the interaction (and most likely PGC-1 activity as well)between a PGC-1 protein and a molecule (target molecule) with which thePGC-1 protein normally interacts. Examples of such target moleculesinclude proteins in the same signaling path as the PGC-1 protein, e.g.,proteins which may function upstream (including both stimulators andinhibitors of activity) or downstream of the PGC-1 protein in a pathwayinvolving regulation of body weight, e.g., PPARγ, C/EBPα, nuclearhormone receptors such as the thyroid hormone receptor, the estrogenreceptor, and the retinoic acid receptor, or in a pathway involvinginsulin sensitivity, e.g., PPARγ. Typically, the assays are cell-freeassays which include the steps of combining a PGC-1 protein or abiologically active portion thereof, a PGC-1 target molecule and acandidate/test compound, e.g., under conditions wherein but for thepresence of the candidate compound, the PGC-1 protein or biologicallyactive portion thereof interacts with (e.g., binds to) the targetmolecule, and detecting the formation of a complex which includes thePGC-1 protein and the target molecule or detecting theinteraction/reaction of the PGC-1 protein and the target molecule.Detection of complex formation can include direct quantitation of thecomplex by, for example, measuring inductive effects of the PGC-1protein. A statistically significant change, such as a decrease, in theinteraction of the PGC-1 and target molecule (e.g., in the formation ofa complex between the PGC-1 and the target molecule) in the presence ofa candidate compound (relative to what is detected in the absence of thecandidate compound) is indicative of a modulation (e.g., stimulation orinhibition) of the interaction between the PGC-1 protein and the targetmolecule. Modulation of the formation of complexes between the PGC-1protein and the target molecule can be quantitated using, for example,an immunoassay.

To perform the above drug screening assays, it is desirable toimmobilize either PGC-1 or its target molecule to facilitate separationof complexes from uncomplexed forms of one or both of the proteins, aswell as to accommodate automation of the assay. Interaction (e.g.,binding of) of PGC-1 to a target molecule, in the presence and absenceof a candidate compound, can be accomplished in any vessel suitable forcontaining the reactants. Examples of such vessels include microtitreplates, test tubes, and micro-centrifuge tubes. In one embodiment, afusion polypeptide can be provided which adds a domain that allows thepolypeptide to be bound to a matrix. For example,glutathione-S-transferase/PGC-1 fusion polypeptides can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe cell lysates (e.g. ³⁵S-labeled) and the candidate compound, and themixture incubated under conditions conducive to complex formation (e.g.,at physiological conditions for salt and pH). Following incubation, thebeads are washed to remove any unbound label, and the matrix immobilizedand radiolabel determined directly, or in the supernatant after thecomplexes are dissociated. Alternatively, the complexes can bedissociated from the matrix, separated by SDS-PAGE, and the level ofPGC-1-binding polypeptide found in the bead fraction quantitated fromthe gel using standard electrophoretic techniques.

Other techniques for immobilizing polypeptides on matrices can also beused in the drug screening assays of the invention. For example, eitherPGC-1 or its target molecule can be immobilized utilizing conjugation ofbiotin and streptavidin. Biotinylated PGC-1 molecules can be preparedfrom biotin-NHS (N-hydroxy-succinimide) using techniques well known inthe art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), andimmobilized in the wells of streptavidin-coated 96 well plates (PierceChemical). Alternatively, antibodies reactive with PGC-1 but which donot interfere with binding of the polypeptide to its target molecule canbe derivatized to the wells of the plate, and PGC-1 trapped in the wellsby antibody conjugation. As described above, preparations of a PGC-1-binding polypeptide and a candidate compound are incubated in the PGC-1-presenting wells of the plate, and the amount of complex trapped in thewell can be quantitated. Methods for detecting such complexes, inaddition to those described above for the GST-immobilized complexes,include immunodetection of complexes using antibodies reactive with thePGC-1 target molecule, or which are reactive with PGC-1 polypeptide andcompete with the target molecule; as well as enzyme-linked assays whichrely on detecting an enzymatic activity associated with the targetmolecule.

In yet another embodiment, the invention provides a method foridentifying a compound (e.g., a screening assay) capable of use in thetreatment of a disorder characterized by (or associated with) aberrantor abnormal PGC-1 nucleic acid expression or PGC-1 polypeptide activity.This method typically includes the step of assaying the ability of thecompound or agent to modulate the expression of the PGC-1 nucleic acidor the activity of the PGC-1 protein thereby identifying a compound fortreating a disorder characterized by aberrant or abnormal PGC-1 nucleicacid expression or PGC-1 polypeptide activity. Disorders characterizedby aberrant or abnormal PGC-1 nucleic acid expression or PGC-1 proteinactivity are described herein. Methods for assaying the ability of thecompound or agent to modulate the expression of the PGC-1 nucleic acidor activity of the PGC-1 protein are typically cell-based assays. Forexample, cells which are sensitive to ligands which transduce signalsvia a pathway involving PGC-1 can be induced to overexpress a PGC-1protein in the presence and absence of a candidate compound. Candidatecompounds which produce a statistically significant change inPGC-1-dependent responses (either stimulation or inhibition) can beidentified. In one embodiment, expression of the PGC-1 nucleic acid oractivity of a PGC-1 protein is modulated in cells and the effects ofcandidate compounds on the readout of interest (such as rate of cellproliferation or differentiation) are measured. For example, theexpression of genes which are up-or down-regulated in response to aPGC-1 protein-dependent signal cascade can be assayed. In preferredembodiments, the regulatory regions of such genes, e.g., the 5′ flankingpromoter and enhancer regions, are operably linked to a detectablemarker (such as luciferase) which encodes a gene product that can bereadily detected. Phosphorylation of PGC-1 or PGC-1 target molecules canalso be measured, for example, by immunoblotting.

Alternatively, modulators of PGC-1 nucleic acid expression (e.g.,compounds which can be used to treat a disorder characterized byaberrant or abnormal PGC-1 nucleic acid expression or PGC-1 proteinactivity) can be identified in a method wherein a cell is contacted witha candidate compound and the expression of PGC-1 mRNA or protein in thecell is determined. The level of expression of PGC-1 mRNA or protein inthe presence of the candidate compound is compared to the level ofexpression of PGC-1 mRNA or protein in the absence of the candidatecompound. The candidate compound can then be identified as a modulatorof PGC-1 nucleic acid expression based on this comparison and be used totreat a disorder characterized by aberrant PGC-1 nucleic acidexpression. For example, when expression of PGC-1 mRNA or polypeptide isgreater (statistically significantly greater) in the presence of thecandidate compound than in its absence, the candidate compound isidentified as a stimulator of PGC-1 nucleic acid expression.Alternatively, when PGC-1 nucleic acid expression is less (statisticallysignificantly less) in the presence of the candidate compound than inits absence, the candidate compound is identified as an inhibitor ofPGC-1 nucleic acid expression. The level of PGC-1 nucleic acidexpression in the cells can be determined by methods described hereinfor detecting PGC-1 mRNA or protein.

In yet another aspect of the invention, the PGC-1 proteins can be usedas “bait proteins” in a two-hybrid assay (see, e.g., U.S. Pat. No.5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J.Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact withPGC-1 (“PGC-1-binding proteins” or “PGC-1-bp”) and modulate PGC-1protein activity. Such PGC-1-binding proteins are also likely to beinvolved in the propagation of signals by the PGC-1 proteins as, forexample, upstream or downstream elements of the PGC-1 pathway.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Bartel, P. et al. “Using the Two-Hybrid System toDetect Protein-Protein Interactions” in Cellular Interactions inDevelopment: A Practical Approach, Hartley, D. A. ed. (Oxford UniversityPress, Oxford, 1993) pp. 153-179. Briefly, the assay utilizes twodifferent DNA constructs. In one construct, the gene that codes forPGC-1 is fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedpolypeptide (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming a PGC-1-dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (e.g., LacZ) which is operablylinked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes thepolypeptide which interacts with PGC-1.

Modulators of PGC-1 protein activity and/or PGC-1 nucleic acidexpression identified according to these drug screening assays can beused to treat, for example, weight disorders, e.g. obesity, anddisorders associated with insufficient insulin activity, e.g., diabetes.These methods of treatment include the steps of administering themodulators of PGC-1 protein activity and/or nucleic acid expression,e.g., in a pharmaceutical composition as described in subsection IVabove, to a subject in need of such treatment, e.g., a subject with adisorder described herein.

b. Diagnostic Assays:

The invention further provides a method for detecting the presence ofPGC-1 in a biological sample. The method involves contacting thebiological sample with a compound or an agent capable of detecting PGC-1polypeptide or mRNA such that the presence of PGC-1 is detected in thebiological sample. A preferred agent for detecting PGC-1 mRNA is alabeled or labelable nucleic acid probe capable of hybridizing to PGC-1mRNA. The nucleic acid probe can be, for example, the full-length PGC-1cDNA of SEQ ID NO:1, or a portion thereof, such as an oligonucleotide ofat least 15, 30, 50, 100, 250 or 500 nucleotides in length andsufficient to specifically hybridize under stringent conditions to PGC-1mRNA. A preferred agent for detecting PGC-1 protein is a labeled orlabelable antibody capable of binding to PGC-1 protein. Antibodies canbe polyclonal, or more preferably, monoclonal. An intact antibody, or afragment thereof (e.g., Fab or F(ab′)₂) can be used. The term “labeledor labelable”, with regard to the probe or antibody, is intended toencompass direct labeling of the probe or antibody by coupling (i.e.,physically linking) a detectable substance to the probe or antibody, aswell as indirect labeling of the probe or antibody by reactivity withanother reagent that is directly labeled. Examples of indirect labelinginclude detection of a primary antibody using a fluorescently labeledsecondary antibody and end-labeling of a DNA probe with biotin such thatit can be detected with fluorescently labeled streptavidin. The term“biological sample” is intended to include tissues, cells and biologicalfluids isolated from a subject, as well as tissues, cells and fluidspresent within a subject. That is, the detection method of the inventioncan be used to detect PGC-1 mRNA or protein in a biological sample invitro as well as in vivo. For example, in vitro techniques for detectionof PGC-1 mRNA include Northern hybridizations and in situhybridizations. In vitro techniques for detection of PGC-1 proteininclude enzyme linked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence. Alternatively, PGC-1protein can be detected in vivo in a subject by introducing into thesubject a labeled anti-PGC-1 antibody. For example, the antibody can belabeled with a radioactive marker whose presence and location in asubject can be detected by standard imaging techniques.

The invention also encompasses kits for detecting the presence of PGC-1in a biological sample. For example, the kit can comprise a labeled orlabelable compound or agent capable of detecting PGC-1 protein or mRNAin a biological sample; means for determining the amount of PGC-1 in thesample; and means for comparing the amount of PGC-1 in the sample with astandard. The compound or agent can be packaged in a suitable container.The kit can further comprise instructions for using the kit to detectPGC-1 mRNA or protein.

The methods of the invention can also be used to detect genetic lesionsin a PGC-1 gene, thereby determining if a subject with the lesioned geneis at risk for a disorder characterized by aberrant or abnormal PGC-1nucleic acid expression or PGC-1 protein activity as defined herein. Inpreferred embodiments, the methods include detecting, in a sample ofcells from the subject, the presence or absence of a genetic lesioncharacterized by at least one of an alteration affecting the integrityof a gene encoding a PGC-1 protein, or the misexpression of the PGC-1gene. For example, such genetic lesions can be detected by ascertainingthe existence of at least one of 1) a deletion of one or morenucleotides from a PGC-1 gene; 2) an addition of one or more nucleotidesto a PGC-1 gene; 3) a substitution of one or more nucleotides of a PGC-1gene, 4) a chromosomal rearrangement of a PGC-1 gene; 5) an alterationin the level of a messenger RNA transcript of a PGC-1 gene, 6) aberrantmodification of a PGC-1 gene, such as of the methylation pattern of thegenomic DNA, 7) the presence of a non-wild type splicing pattern of amessenger RNA transcript of a PGC-1 gene, 8) a non-wild type level of aPGC-1-protein, 9) allelic loss of a PGC-1 gene, and 10) inappropriatepost-translational modification of a PGC-1-protein. As described herein,there are a large number of assay techniques known in the art which canbe used for detecting lesions in a PGC-1 gene.

In certain embodiments, detection of the lesion involves the use of aprobe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Pat.Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) PNAS91:360-364), the latter of which can be particularly useful fordetecting point mutations in the PGC-1-gene (see Abravaya et al. (1995)Nucleic Acids Res. 23:675-682). This method can include the steps ofcollecting a sample or cells from a patient, isolating nucleic acid(e.g., genomic, mRNA or both) from the cells of the sample, contactingthe nucleic acid sample with one or more primers which specificallyhybridize to a PGC-1 gene under conditions such that hybridization andamplification of the PGC-1-gene (if present) occurs, and detecting thepresence or absence of an amplification product, or detecting the sizeof the amplification product and comparing the length to a controlsample.

In an alternative embodiment, mutations in a PGC-1 gene from a samplecell can be identified by alterations in restriction enzyme cleavagepatterns. For example, sample and control DNA is isolated, amplified(optionally), digested with one or more restriction endonucleases, andfragment length sizes are determined by gel electrophoresis andcompared. Differences in fragment length sizes between sample andcontrol DNA indicates mutations in the sample DNA. Moreover, the use ofsequence specific ribozymes (see, for example, U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the PGC-1 gene anddetect mutations by comparing the sequence of the sample PGC-1 with thecorresponding wild-type (control) sequence. Examples of sequencingreactions include those based on techniques developed by Maxim andGilbert ((1977) PNAS 74:560) or Sanger ((1977) PNAS 74:5463). A varietyof automated sequencing procedures can be utilized when performing thediagnostic assays ((1995) Biotechniques 19:448), including sequencing bymass spectrometry (see, e.g., PCT International Publication No. WO94/16101; Cohen et al. (1996) Adv. Chromatogr. 36:127-162; and Griffinet al. (1993) Appl. Biochem. Biotechnol. 38:147-159).

Other methods for detecting mutations in the PGC-1 gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA duplexes (Myers et al. (1985) Science230:1242); Cotton et al. (1988) PNAS 85:4397; Saleeba et al. (1992)Meth. Enzymol. 217:286-295), electrophoretic mobility of mutant and wildtype nucleic acid is compared (Orita et al. (1989) PNAS 86:2766; Cotton(1993) Mutat Res 285:125-144; and Hayashi (1992) Genet Anal Tech Appl9:73-79), and movement of mutant or wild-type fragments inpolyacrylamide gels containing a gradient of denaturant is assayed usingdenaturing gradient gel electrophoresis (Myers et al (1985) Nature313:495). Examples of other techniques for detecting point mutationsinclude, selective oligonucleotide hybridization, selectiveamplification, and selective primer extension.

c. Methods of Treatment

Another aspect of the invention pertains to methods for treating asubject, e.g., a human, having a disease or disorder characterized by(or associated with) aberrant or abnormal PGC-1 nucleic acid expressionand/or PGC-1 protein activity. These methods include the step ofadministering a PGC-1 modulator to the subject such that treatmentoccurs. The language “aberrant or abnormal PGC-1 expression” refers toexpression of a non-wild-type PGC-1 protein or a non-wild-type level ofexpression of a PGC-1 protein. Aberrant or abnormal PGC-1 proteinactivity refers to a non-wild-type PGC-1 protein activity or anon-wild-type level of PGC-1 protein activity. As the PGC-1 protein isinvolved in, for example, a pathway involving adipocyte differentiation,thermogenesis in brown adipocytes, and insulin sensitivity, aberrant orabnormal PGC-1 protein activity or nucleic acid expression interfereswith the normal weight control and metabolic functions. Non-limitingexamples of disorders or diseases characterized by or associated withabnormal or aberrant PGC-1 protein activity or nucleic acid expressioninclude weight disorders, e.g., obesity, cachexia, anorexia, anddisorders associated with insufficient insulin activity, e.g., diabetes.Disorders associated with body weight are disorders associated withabnormal body weight or abnormal control of body weight. As used herein,the language “diseases associated with or characterized by insufficientinsulin activity” include disorders or diseases in which there is anabnormal utilization of glucose due to abnormal insulin function.Abnormal insulin function includes any abnormality or impairment ininsulin production, e.g., expression and/or transport through cellularorganelles, such as insulin deficiency resulting from, for example, lossof β cells as in IDDM (Type I diabetes), secretion, such as impairmentof insulin secretory responses as in NIDDM (Type 11 diabetes), the formof the insulin molecule itself, e.g., primary, secondary or tertiarystructure, effects of insulin on target cells, e.g., insulin-resistancein bodily tissues, e.g., peripheral tissues, and responses of targetcells to insulin. See Braunwald, E. et al. eds. Harrison's Principles ofInternal Medicine, Eleventh Edition (McGraw-Hill Book Company, New York,1987) pp. 1778-1797; Robbins, S. L. et al. Pathologic Basis of Disease,3rd Edition (W. B. Saunders Company, Philadelphia, 1984) p. 972 forfurther descriptions of abnormal insulin activity in IDDM and NIDDM andother forms of diabetes. The terms “treating” or “treatment”, as usedherein, refer to reduction or alleviation of at least one adverse effector symptom of a disorder or disease, e.g., a disorder or diseasecharacterized by or associated with abnormal or aberrant PGC-1 proteinactivity or PGC-1 nucleic acid expression.

As used herein, a PGC-1 modulator is a molecule which can modulate PGC-1nucleic acid expression and/or PGC-1 protein activity. For example, aPGC-1 modulator can modulate, e.g., upregulate (activate) ordownregulate (suppress), PGC-1 nucleic acid expression. In anotherexample, a PGC-1 modulator can modulate (e.g., stimulate or inhibit)PGC-1 protein activity. If it is desirable to treat a disorder ordisease characterized by (or associated with) aberrant or abnormal(non-wild-type) PGC-1 nucleic acid expression and/or PGC-1 proteinactivity by inhibiting PGC-1 nucleic acid expression, a PGC-1 modulatorcan be an antisense molecule, e.g., a ribozyme, as described herein.Examples of antisense molecules which can be used to inhibit PGC-1nucleic acid expression include antisense molecules which arecomplementary to a portion of the 5′ untranslated region of SEQ ID NO:1,SEQ ID NO:4 which also includes the start codon and antisense moleculeswhich are complementary to a portion of the 3′ untranslated region ofSEQ ID NO:1, SEQ ID NO:4. A PGC-1 modulator which inhibits PGC-1 nucleicacid expression can also be a small molecule or other drug, e.g., asmall molecule or drug identified using the screening assays describedherein, which inhibits PGC-1 nucleic acid expression. If it is desirableto treat a disease or disorder characterized by (or associated with)aberrant or abnormal (non-wild-type) PGC-1 nucleic acid expressionand/or PGC-1 protein activity by stimulating PGC-1 nucleic acidexpression, a PGC-1 modulator can be, for example, a nucleic acidmolecule encoding PGC-1 (e.g., a nucleic acid molecule comprising anucleotide sequence homologous to the nucleotide sequence of SEQ IDNO:1, SEQ ID NO:4) or a small molecule or other drug, e.g., a smallmolecule (peptide) or drug identified using the screening assaysdescribed herein, which stimulates PGC-1 nucleic acid expression.

Alternatively, if it is desirable to treat a disease or disordercharacterized by (or associated with) aberrant or abnormal(non-wild-type) PGC-1 nucleic acid expression and/or PGC-1 proteinactivity by inhibiting PGC-1 protein activity, a PGC-1 modulator can bean anti-PGC-1 antibody or a small molecule or other drug, e.g., a smallmolecule or drug identified using the screening assays described herein,which inhibits PGC-1 protein activity. If it is desirable to treat adisease or disorder characterized by (or associated with) aberrant orabnormal (non-wild-type) PGC-1 nucleic acid expression and/or PGC-1protein activity by stimulating PGC-1 protein activity, a PGC-1modulator can be an active PGC-1 protein or portion thereof (e.g., aPGC-1 protein or portion thereof having an amino acid sequence which ishomologous to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:5 or aportion thereof) or a small molecule or other drug, e.g., a smallmolecule or drug identified using the screening assays described herein,which stimulates PGC-1 protein activity.

In addition, a subject having a weight disorder, e.g., obesity, can betreated according to the present invention by administering to thesubject a PGC-1 protein or portion thereof or a nucleic acid encoding aPGC-1 protein or portion thereof such that treatment occurs. Similarly,a subject having a disorder associated with insufficient insulinactivity can be treated according to the present invention byadministering to the subject a PGC-1 protein or portion thereof or anucleic acid encoding a PGC-1 protein or portion thereof such thattreatment occurs.

Other aspects of the invention pertain to methods for modulating a cellassociated activity. These methods include contacting the cell with anagent (or a composition which includes an effective amount of an agent)which modulates PGC-1 protein activity or PGC-1 nucleic acid expressionsuch that a cell associated activity is altered relative to a cellassociated activity of the cell in the absence of the agent. As usedherein, “a cell associated activity” refers to a normal or abnormalactivity or function of a cell. Examples of cell associated activitiesinclude proliferation, migration, differentiation, production orsecretion of molecules, such as proteins, cell survival, andthermogenesis. In a preferred embodiment, the cell associated activityis thermogenesis and the cell is a brown adipocyte. The term “altered”as used herein refers to a change, e.g., an increase or decrease, of acell associated activity. In one embodiment, the agent stimulates PGC-1protein activity or PGC-1 nucleic acid expression. Examples of suchstimulatory agents include an active PGC-1 protein, a nucleic acidmolecule encoding PGC-1 that has been introduced into the cell, and amodulatory agent which stimulates PGC-1 protein activity or PGC-1nucleic acid expression and which is identified using the drug screeningassays described herein. In another embodiment, the agent inhibits PGC-1protein activity or PGC-1 nucleic acid expression. Examples of suchinhibitory agents include an antisense PGC-1 nucleic acid molecule, ananti-PGC-1 antibody, and a modulatory agent which inhibits PGC-1 proteinactivity or PGC-1 nucleic acid expression and which is identified usingthe drug screening assays described herein. These modulatory methods canbe performed in vitro (e.g., by culturing the cell with the agent) or,alternatively, in vivo (e.g., by administering the agent to a subject).In a preferred embodiment, the modulatory methods are performed in vivo,i.e., the cell is present within a subject, e.g., a mammal, e.g., ahuman, and the subject has a disorder or disease characterized by orassociated with abnormal or aberrant PGC-1 protein activity or PGC-1nucleic acid expression.

A nucleic acid molecule, a protein, a PGC-1 modulator, a compound etc.used in the methods of treatment can be incorporated into an appropriatepharmaceutical composition described herein and administered to thesubject through a route which allows the molecule, protein, modulator,or compound etc. to perform its intended function. Examples of routes ofadministration are also described herein under subsection IV.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patent applications, patents, and published patent applications citedthroughout this application are hereby incorporated by reference.

EXAMPLES Example I Identification and Characterization of Mouse PGC-1

The mouse HIB 1B cell line (Ross, R. et al. (1992) Proc. Natl. Acad.Sci. USA 89:7561-7565), a brown adipocyte cell line which expresses UCP,was differentiated and treated with isoproterenol to induce UCPexpression. A cDNA library from the mouse HIB 1B cell line was screenedin a yeast two hybrid system using PPARγ as bait and Clontech (PaloAlto, Calif.) reagents. Briefly, amino acids 183-505 of the murine PPARγwere cloned in-frame into the GAL4 DNA-binding domain plasmid pAS2. AHIB 1B cDNA expression library was constructed in the GAL4 activationdomain plasmid pACT II. The yeast two-hybrid system protocol wasperformed as described in the CLONTECH Matchmaker two-hybrid systemprotocol. pAS-PPARγ was transformed into Y190 yeast cells by the lithiumacetate method and maintained by selection in leucine-plates. A pACT-HIB1B cDNA library was transformed into Y190-PPARγ yeast cells, andpositive clones were assayed for β-galactosidase activity in a filterassay as described in the CLONTECH protocol. pAS1 lamin cDNA was used toobtain full-length PGC-1, the positive yeast cDNA clone was used asprobe to screen an oligo dT λZAP cDNA library from HIB 1B cells.

A screen of 1×10⁶ primary transformants using cDNAs prepared from HIB 1Bbrown fat cells yielded about 130 clones. The cDNA inserts of positivephage clones were excised into pBluescript and both strands weresequenced by standard methods. These were then analyzed for preferentialexpression in brown versus white fat with RNA blots. One of the clonesobtained using this yeast two hybrid system was a partial PGC-1 clonewhich comprised nucleotides 610 to 3066 of SEQ ID NO:1. The full lengthclone was obtained by using a partial PGC-1 clone comprising nucleotides650 to 3066 of SEQ ID NO:1, to screen a λZAP-HIB 1B library.

PGC-1 was then subcloned from a PBS plasmid to a PSV sport (GIBCO BRL,Gaithersburg, Md.) and in vitro translated using the TnT Promega kit(Promega, Madison, Wis.). Two bands were observed in the in vitrotranslated PSV sport PGC-1 which corresponded to the molecular weightsof about 120 kD and 70 kD. These bands most likely represent differentisoforms of PGC-1 . The 120 kD form most likely represents the proteinof SEQ ID NO:2.

The nucleotide sequence of murine PGC-1 (shown in FIGS. 1A, 1B, and 1Cand SEQ ID NO:1) includes 3066 nucleotides which encode a proteincontaining 797 amino acid residues with a predicted molecular mass of 92kDa (FIG. 2A). The murine PGC-1 protein sequence (shown in FIGS. 1A, 1B,1C, and 2A and SEQ ID NO:2) has several domains/motifs includingDatabank searches indicate that murine PGC-1 represents, a novel proteinwith no close homologs in any databases except expressed sequence tag(EST) databases. It does, however, contain recognizable peptide motifsincluding: a putative RNA-binding motif (amino acids 677-709) and twoso-called SR domains, regions that are rich in serine and arginineresidues (amino acids 565-598 and 617-631). Proteins containing pairedRNA-binding motifs and SR domains have been shown to interact with theC-terminal domain (CTD) of RNA polymerase II (Yuryev et al. (1996) Proc.Natl. Acad. Sci. USA 93:6975-6980). Except for these two regions,however, PGC-1 shares no other sequence similarity with other proteinsthat contain these domains. In addition to these domains, PGC-1 alsocontains three consensus sites for phosphorylation by protein kinase A.However, no significant homology was discovered between PGC-1 and anyknown coactivator of nuclear receptors. PGC-1 does, however, contain oneLXXLL motif (amino acids 142-146), recently identified as an elementthat can mediate nuclear receptor-coactivator interactions (Heery et al.(1997) Nature 397:733-736; Torchia et al. (1997) Nature 387:677-684).

From these experiments, it is clear that PGC-1 is a new factor thatinteracts with the adipogenic transcription factor PPARγ. Moreover, asit is known that ligands of PPARγ can induce the specific brown adiposetissue marker UCP, PPARγ is believed to play an important role in brownadipose tissue differentiation. Thus, PGC-1 modulation of PPARγ activityplays a role in brown adipose tissue differentiation, e.g., it canpromote cells to differentiate into brown adipose cells rather thanwhite adipose cells.

Example II Identification of Human PGC-1

Northern blot analysis of human poly A RNA screened with a mouse fulllength cDNA probe (e.g., a probe having the sequence shown in SEQ IDNO:1) revealed high levels of expression of PGC-1 in human muscle,heart, brain, kidney and pancreas, with the highest levels of expressiondetected in human muscle and heart. Accordingly, a human muscle library,e.g., an human skeletal muscle oligo dT primed library (Clontech catalog#HL5023t, lot # 7110299), was screened using a full length mouse PGC-1probe comprising the nucleotide sequence of SEQ ID NO:1 or a portionthereof (e.g., nucleotides from the 5′ region of SEQ ID NO:1, e.g.,nucleotides 1-50 of SEQ ID NO:1) (e.g., as described in Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989). Several overlapping clones wereisolated and sequenced. After several rounds of screening, the longestclone isolated (clone #1¹) contained a fragment with homology startingat amino acid 507 of the mouse sequence in SEQ ID NO:1.

Using a “5′ race strategy” the full length cDNA sequence was obtained. Ahuman PGC-1 cDNA clone was obtained with the Marathon RACE protocol andreagents available commercially through Clontech Laboratories, Inc.RACE, or rapid amplification of cDNA ends, is useful to isolate a PCRfragment comprising the native 3′ or 5′ end of a cDNA open readingframe, and involves use of one or more gene-specific sense (for 3′ RACE)or antisense (for 5′ RACE) oligonucleotide primers. The RACE protocolused is generally as described in Siebert et al. (1995), 23 Nucl. AcidsRes. 1087-1088, and in the Clontech, Inc. User Manual for Marathon-ReadycDNA (1996), the teachings of which are incorporated herein byreference. The RACE reagents included the Advantage KlenTaq Polymerasemix, 10X PCR reaction buffer, 50X dNTP mix and Tricine-EDTA buffercommercially available from Clontech, Inc. The protocol is practicedwith 0.5 mL PCR reaction tubes and a thermal cycling device such as theDNA Thermal Cycler 480 available from Perkin-Elmer Corporation.

A PGC-1 specific primer (ATCTTCGCTGTCATCAAACAGGCCATC (SEQ ID NO:6)), 27bp (base-pairs) in length, was prepared for use in a 5′-RACE protocol toamplify a PCR product comprising the 5′ end of human PGC-1 open readingframe in a Human Skeletal Muscle Marathon-Ready cDNA preparation(Clontech catalog # 7413-1, lot # 8030061). Thermal cycling was carriedout according to the manufacturer's recommended Program 1 (a 94° C. hotstart followed by 5 cycles at 94° C. to 72° C., then 5 cycles at 94° C.to 70° C., then 20-25 cycles at 94° C. to 68° C.). Confirmation thatadditional human PGC-1 gene sequence has been obtained can be producedby routine Southern blot analysis or by subcloning and sequencing. Thisfragment contained a sequence with homology extending to the most 5′sequence of mouse PGC-1 (SEQ ID NO:1). Both clone # 1¹ and the 5′ RACEproduct were then sequenced on both strands and the full length humancDNA sequence (SEQ ID NO:4) was constituted.

The nucleotide sequence of human PGC-1 (shown in FIGS. 7A and 7B and SEQID NO:4) includes 3023 nucleotides which encode a protein containing 798amino acid residues with a predicted molecular mass of 92 kDa. The humanPGC-1 protein sequence (shown in FIG. 8 and SEQ ID NO:5) has severaldomains/motifs including Databank searches indicate that human PGC-1represents a novel protein with no close homologs in any databasesexcept expressed sequence tag (EST) databases.

An alignment between human PGC-1 (SEQ ID NO:5) and mouse PGC-1 (SEQ IDNO:2) amino acid sequences was performed using the BLAST software foundat the National Center for Biotechnology Information (NCBI) web site(URL: http://www.ncbi.nlm.nih.gov, Altschul, S. F. et al. (1990) J. MolBiol 215:403-410; Madden, T. L. et al. (1996) Meth Enzymol 266:131-141)and it was determined that human PGC-1 has a 94% identity to mousePGC-1.

Example III Tissue Distribution of Mouse PGC-1 and Cold Inducation ofPGC-1 in Brown Adipose Tissue

A Northern analysis of mRNA from lung, muscle, liver, heart, kidney,white adipose tissue (WAT), brown adipose tissue (BAT), brain, testis,and spleen tissue from 4 week old mice acclimated at 24° C. using aprobe comprising nucleotides 150 to 3066 of SEQ ID NO:1 was performed.Briefly, total RNA was isolated from cultured cells and tissues of mouseby guanidine isothiocyanate extraction. RNA samples were processed aspreviously described (Tontonoz et al. (1994) Genes Dev. 8:1224-1234).Three bands appeared on the Northern blots that were larger than the 28S(5000-6000 bp) marker. These bands most likely represent differentisoforms of PGC-1. PGC-1 mRNA was detected predominantly in brain,heart, kidney, and BAT. In addition, a minor species of approximately 8kb is also observed in all of these tissues. In contrast, no PGC-1 mRNAexpression is observed from white fat, lung, skeletal muscle, liver,testes, or spleen.

Exposure to cold is a classical inducer of adaptive thermogenesis,especially in brown fat and skeletal muscle (Himms-Hagen (1989) Can. JPhysiol. Pharmacol. 67:394-401). A second Northern analysis of mRNA fromWAT, BAT, and liver tissue from 4 week old mice acclimated at 4° C. from3 to 12 hours using the same probe as in the first Northern analysis wasperformed. From this Northern analysis, it was apparent that PGC-1 washighly induced (about 30-to 50-fold) during cold exposure especially inBAT and that PGC-1 expression was BAT specific with no expression inWAT. Although PGC-1 mRNA expression is not detectable in skeletal musclefrom mice kept at ambient temperature, exposure of mice to cold for 12hr induces expression of the PGC-1 gene in this tissue. Heart andkidney, which express PGC-1 mRNA at room temperature, do not elevatethis expression upon cold exposure. PGC-1 induction during cold exposureparallels that of UCP, a brown fat specific marker responsible for thethermogenic activity of BAT.

These experiments show that although PGC-1 is -expressed in severaltissues, including BAT, from animals acclimated to 24° C., it is notexpressed in WAT. The animal studies described herein were carried outas follows. Four-week-old male C57BL/6J mice were used. Animals were fedad libitum and 10 animals were grouped per cage. A control group waskept at 24° C., while experimental groups were kept at 4° C. for 3 or 12hrs. Animals were sacrificed, tissues were dissected and collectedimmediately.

WAT and BAT share the same genetic and biochemical machinery foradipogenesis except that BAT develops a thermogenic function uponterminal differentiation. Thus, PGC-1 plays a role in the thermogenicfunction of BAT. This function was confirmed when the second Northernanalysis revealed that in tissues from animals acclimated at 4° C.,PGC-1 was expressed essentially only in BAT. PGC-1, therefore, plays arole in the equilibrium between energy storage and expenditure.

Northern blot mRNA analysis of PGC-1 and genes of mitochondrial functionin different mouse tissues (kidney, heart, BAT and WAT) after coldexposure revealed that cold-induced expression of PGC-1 in the brown fatof these mice correlated with the induced expression of other keymitochondrial proteins including ATP-synthetase (β subunit) andcytochrome c-oxidase subunits (COX II and COX IP). Although chronic coldexposure has been reported to lead to elevated activities for thesemitochondrial proteins in skeletal muscle (Bourhim et al. (1990) Am. JPhysiol. 258:R1291-R1298), no induction of mRNA for ATP-synthetase, COXII or COX IV was seen in muscle with the relatively brief exposure tocold. To conduct these experiments, animal were maintained at 4° C. for3 or 12 hours, sacrificed and tissues (kidney, heart, WAT and BAT) weredissected for the preparation of RNA. Ten mice were pooled for eachsample. Probes used for hybridization were PGC-1, UCP-1, ATP synthetase(β subunit), cytochrome c-oxidase II (COX-II), and cytochrome c-oxidaseIV (COX-IV).

Cold is sensed in the central nervous system and results in increasedsympathetic output to peripheral tissues, including muscle and brown fatHimms-Hagen (1989) Can. J. Physiol. Pharmacol. 67:394-401). Coldexposure can be mimicked, in terms of brown adipocyte precursor cellgrowth and the induction of UCP-1, by exposure of cultured brown fatcells to β-adrenergic agonists (Rehnmark et al. (1990) J. Biol. Chem.25:16464-16471). To determine if PGC-1 gene expression is also sensitiveto β-adrenergic agonists, HIB IB brown fat cells were treated withisoproterenol (1 μM), a nonsubtype selective β agonist, for 10 hr. Totalcellular RNA was isolated and analyzed using PGC-1 and UCP-1 cDNAprobes.

Treatment of HIB 1B brown fat cells with these agents resulted in asharp increase in both PGC-1 mRNA and UCP-1 mRNA. Briefly, HIB 1B brownfat preadipocytes were differentiated as described herein. After 6 days,cells were approximately 80% differentiated. Exposure of brown fat cellsto 9-cis retinoic acid has previously been shown to potentiate theeffects of β agonists to induce UCP-1 expression (Puigserver et al.(1996) Biochem. J. 317:827-833). Addition of this retinoid (whichactivates both RXR and RAR) and isoproterenol to the HIB 1B cellsresulted in a small, further increase in both PGC-1 and UCP-Iexpression. These results indicate that β-adrenergic agonists may playan important role in mediating the effects of cold on the induction ofboth UCP-1 and PGC-1.

Example IV Recombinant Expression of PGC-1 and Binding of PGC-1 to otherTranscription Factors and Nuclear Hormone Receptors

GST-PGC-1 fusion proteins were generated by first subcloning a portionof the PGC-1 nucleotide sequence (nucleotides 610 to 3066 of SEQ IDNO:1) into a pGEX vector (Pharmacia Biotech Inc., Piscataway, N.J.).Briefly, PGC-1 (EcoRI-Xhol fragment from pBluescript) was cloned intothe Smal site of pGEX 5X3. The PPARγ deletions were generated byperforming PCR using specific oligonucleotides and there were clonedin-frame in pGEX 5X2. These fusion proteins were expressed, and purifiedfrom E. coli on beads containing approximately 1 μg of protein (eitherGST, or alone, or fused to PGC-1), 30 μl was resuspended i the bindingbuffer (20 mM HEPES [pH 7.7], 75 mM KCl, 0.1 mM EDTA, 2.5 mM MgCl2,0.05% NP40,2 mM DTT, 10% glycerol).

After expressing the fusion protein in COS cells, in vitro bindingassays as described in Takeshita, A. et al. ((1996) Endocrinology137:3594-3597) were performed to study the interaction of PGC-1 withPPARγ, other PPAR isoforms such as PPARα and PPARδ, other transcriptionfactors such as C/EBPα and RXRα, and other nuclear hormone receptorssuch as the thyroid hormone receptor, the estrogen receptor, and theretinoic acid receptor. The assays were carried out as follows. ControlGST protein alone or PGC-1 (aa 36-797) fused to GST were immobilized onglutathione agarose beads and incubated with different invitro-translated ([³⁵S]methionine-labeled nuclear receptors andappropriate ligands or vehicle. The fusion proteins were mixed with 5 μlof different nuclear receptors made in an in vitro reticulocytetranslation reaction using [³⁵S] methionine (Promega TNT reticulocytelysate system kit). Specific nuclear receptor ligands or vehicle (5 μl)was added. Binding was performed for 60 min. at room temperature. Thebeads were then washed four times with the binding buffer with orwithout ligands and resuspended in SDS-PAGE sample buffer. Afterelectrophoresis, fixation, and enhancement, the radiolabeled proteinswere visualized by autoradiography.

These assays show that PGC-1 interacted with PPARγ. This interaction wasnot ligand-dependent, in that addition of BRL49653 (a thiazolidinedioneligand for PPARγ) at 10 μM does not significantly alter this binding. Asimilar lack of ligand dependence for this interaction was seen whenbacterially expressed PPARγ was immobilized on beads and used withreticulocyte-translated PGC-1. These assays also showed that PGC-1interacts with: a) PPARα and shows a slight ligand dependency usingleukotriene-4 (1 μM); b) PPARδ and shows a slight ligand dependencyusing carboprostacyclin (1 μM); c) the thyroid hormone receptor with aslight ligand dependency using thyroid hormone (1 μM); d) the estrogenreceptor with a slight ligand dependency using estradiol (1 μM); and 3)the retinoic acid receptor with a strong ligand dependency usingall-trans retinoic acid (1 μM). The TRβ also binds specifically toPGC-1, though in this case ligand (T₃) addition causes a 2-to 3-foldincrease in binding. A strong ligand-dependent binding is seen betweenPGC-1 and the retinoic acid (RA) receptor, and between PGC-1 and theestrogen receptor (ERα). In contrast, little or no binding is seenbetween PGC-1 and the retinoid-X receptor (RXRα), with or without ligandaddition. These data indicate that PGC-1 interacts specifically withPPARγ and several other nuclear receptors in vitro. There is a broadrange of dependence on ligand for these interactions, from no liganddependence (PPARγ) to a strong dependence on ligand addition (RARα).

The interaction between PPARγ and PGC-1 can also be seen in mammaliancells. Even in the absence of added ligand, an association is observedbetween these two proteins in immunoprecipitation assays. Vectorsexpressing HA-tagged PGC-1 and PPARγ were transfected into COS cells. Inbrief, full-length PGC-1 with an HA-tagged N terminus was generated byPCR and closed into Smal of pSV-SPORT. Ligands pioglitazone (5 μM),9-cis RA (1 μM), and 8-Br-cAMP (1 nm) were added 3 hrs. before cellswere harvested. Cell extracts and immunoprecipitation from transfectedcells were performed as in Lassar et al. ((1991) Cell 66:305-315).Rabbit anti-murine PPARγ (Hu et al. (1996) Science 274:2100-2103) wasused as a 1:500 dilution for immunoprecipitation. An anti-HA mousedilution for Western blot that was developed using ECL (Amersham). Whencells are treated with pioglitazone (a PPARγ ligand), a very modestincrease in association is observed.

To address whether PGC-1 does indeed reside in the cell nucleus, afusion protein between PGC-1 and green fluorescent protein (GFP) wasconstructed. GFP fused to the full-length PGC-1 was generated by closingthis (Clontech). Cellular localization was visualized 24 hrs. aftertransfection using a Nikon Diaphora 200 microscope. When GFP-PGC-1 isexpressed in COS cells, it is observed entirely in the cell nucleus.

These results show that PGC-1 binds not only to PPARγ but also to othernuclear hormone receptors, and thus this molecule can be used tomodulate the function of these additional nuclear hormone receptors.PGC-1 can be used as a target for screening molecules which modulate thefunction of these nuclear hormone receptors. Moreover, the fact thatPGC-1 interacts with the thyroid hormone receptor and the retinoic acidreceptor is important in brown adipocyte function as both of thesereceptors can transcriptionally regulate UCP expression.

Example V PGC-1 Acts as a Coactivator with PPARγ/PXRα and TR to induceExpression of a Gene Under The Control of UCP Regulartory Elements

To assess the transcription activity of PGC-1, an in vitrotranscriptional assay was performed. The UCP-1 promoter has been shownto have binding sites for both PPARγ and the TR (Cassard-Doulcier et al.(1994) J. Biol. Chem. 269:24335-24342; Sears et al. (1996) Mol. Cell.Biol. 16:3410-3419). In this assay, the full length promoter andenhancer of UCP was linked to the CAT reporter gene. RAT IR (a ratfibroblast cell line transformed to express the human insulin receptor)cells were transiently transfected with PSV-sport alone (control),PPARγ/RXRα, PGC-1, and PPARγ/RXRα/PGC-1 using the calcium phosphatemethod. Results from CAT assays were controlled for transfectionefficiency by cotransfection of a β-galactosidase reporter gene underthe control of the CMV promoter. In each case, the cells were treatedwith either dimethyl sulfoxide or a combination of 9-cis retinoic acid,8-Br-cAMP, and the synthetic PPARγ ligand pioglitazone (PIO).Transcriptional activity was seen in the cells treated with thecombination of 9-cis retinoic acid, 8-Br-cAMP, and PIO and containingPGC-1 alone, cells containing PPARγ/RXRα, and cells containingPPARγ/RXRα/PGC-1. Maximum activity was seen in cells treated with thecombination of 9-cis retinoic acid, 8-Br-cAMP, and PIO and containingPPARγ/RXRα/PGC-1. These results indicate that PGC-1 acts as a positivetranscriptional coactivator of PPARγ/RXRα.

To determine which inducers were involved in the transcriptionalactivation of PGC-1, the cells were treated individually (PIO, thesynthetic PPARγ ligand troglitazone (TRO), 9-cis retinoic acid, 8-BrcAMP) and in combination (9-cis retinoic acid in combination with 8-BrcAMP) with different inducers. With regard to the cells treated with theindividual inducers, it was found that the potency of the inducers wasas follows (from highest to lowest): 9-cis retinoic acid, 8-Br cAMP,TRO, and then PIO. The combination of 9-cis retinoic acid and 8-Br cAMPwas more potent in enhancing transcriptional activity than any of theindividual inducers.

Similarly, TRβ/RXRα combination alone induced very littletranscriptional activity, even when stimulated with a ligand cocktailincluding T₃ (1 μM). However, the combination of PGC-1 with the TRβ/RXRαpair induced powerful transactivation, again in a ligand-dependentmanner. These results clearly indicate that PGC-1 can function as apotent transcriptional coactivator for PPARγ and the TR. It isinteresting that the optimal transcriptional response is seen with PPARγligand is added, despite the fact that the binding of PGC-1 and PPARγ isnot ligand dependent. It is likely that this results from simultaneous,ligand-dependent docking of another coactivator, such as SRC-1, CBP, orothers.

The role of different hormones and ligands used to achieve maximaltranscriptional activation with PPARγ and PGC-1 is dissected in FIG. 3A.The individual components -troglitazone (trog.), 9 cis-retinoic acid(9cRA), and 8-bromo cyclic AMP (cAMP) -each stimulate a 2-to 4-foldincrease in transcriptional activity. The fold activation was comparedto the value observed in cells transfected with the same vectors but nottreated with ligand. However, the most robust responses are seen whenthey are used in combination. The synergistic effect of 9-cis retinoicacid and 8-bromo cyclic AMP is particularly striking (14-fold), whileall three agents together cause an 18-fold increase above the untreatedcontrol.

The above-described transcriptional assay represents a useful assay forscreening compounds or agents which can modulate, e.g., stimulate orinhibit, the function of PGC-1 alone and/or PGC-1 in combination withPPARγ/RXRα. Based on the results reported in this Example, agents whichlikely modulate UCP expression and thus thermogenesis in BAT includePGC-1 molecules, PPARγ ligands (e.g., thiazolidinediones, e.g., PIO andTRO), retinoids, and adrenergic agonists.

Example VI Identification of the Domains that Mediate the PGC-1-PPARγInteraction

The interaction between nuclear receptors and certain coactivators suchas SRC-1 or CBP is ligand dependent (Kamai et al. (1996) Cell84:403-414) and involves an LXXLL (SEQ ID NO:3) motif in thecoactivators and the C-terminal AF-2 domain in the receptors (Heery etal. (1997) Nature 387:733-736; Torchia et al. (1997) Nature387:677-684). To identify the domains responsible for PGC-I-PPARγinteractions, different C-terminal deletions of PGC-1 were generated asreticulocyte translation products and mixed with a FST-PPARγ fusionprotein. Deletions of PGC-1 were made using specific restriction sitesin the PGC-1 were made using specific restriction sites in the PGC-1cDNA closed in pBluescript. The following restriction enzymes were usedfor these deletions: full-length Xhol (aa-1-797), Haell (aa 1-675) Ncol(aa 1-503), Xbal (aa 1-403), Kpnl (aa 1-338), and Stul (aa 1-292). Thesewere then translated into vitro with an [³⁵S]methionine-label. Onemicroliter of each in vitro translation reaction was resolved bySDS-PAGE and autoradiographed.

FIG. 4 summarizes the input of both the full-length PGC-1 (1-797) andthe 1-675 deletion which bind to the immobilized PPARγ. The binding ofPGC-1 1-503, which lacks the SR and RNA-binding domains, is modestlydecreased to 18%. A similar level of binding can be seen for PGC-1 1-403and 1-338. However, PGC-11-292, which still contains the LXXLL (SEQ IDNO:3) motif, completely loses the ability to interact with PPARγ. Asshown in FIG. 2A, residues 292-338 contain no distinct domains known tomediate protein-protein interaction, though it is very rich in prolineresidues.

Most of the nuclear hormone receptor coactivators identified to dateinteract with the C-terminal AF-2 domain, which is responsible forligand-dependent transcriptional activation. To determine if PGC-1 alsointeracts with this part of PPARγ, several deletions of PPARγ preparedas GST fusion proteins were used and combined with in vitro-translatedPGC-1. FIG. 5 shows that amino acids 181-505 of PPARγ (the originalfragment used in the yeast two-hybrid screen) interact strongly withPGC-1, pulling down 23% of the input. On the other hand, a furtherdeletion of 45 amino acids (228-505) is not able to bind to full-lengthPGC-1. Both of these PPARγ deletions were able to bind SRC-1, indicatingthat they have not lost their general ability to interact with otherproteins. These data demonstrate that PPARγ utilizes part of itsDNA-binding and hinge domains to bind PGC-1. It apparently does notinteract through the C-terminal AF-2 domain that docks othercoactivators such as SRC-1 and CBP.

Example VII Transcriptional Activity and Deletion Analysis of PGC-1

To address whether PGC-1 has its own transcriptional activation domainor contains some activity that might unmask or augment thetranscriptional activator properties of the nuclear receptors, a numberof fusion proteins between full length or portions of PGC-1 and theDNA-binding domain (DBD) of GAL4 were prepared and assayed transcriptionthrough a GAL4 DNA binding target sequence, the UAS. Transcription wasassayed with a reporter plasmid containing five copies of the UAS linkedto CAT. More specifically, transcriptional activation assays wereperformed as follows. An expression plasmid containing full-length PGC-1was constructed by first ligating the entire 3 kb cDNA as a Small-Xholfragment into Smal-Sall sites of pSV-SPORT (GIBCO-BRL). This wasexpressed in cells with -CMX vector, along with a control fusion betweenGAL4 DBD and full-length murine SRCl. The activity stimulated by 4.5 μgof the DBD-PGC-1 was set as 100%. The −3740/+110 bp UCP promoter wasdescribed previously (Kozak et al. (1994) Mol. Cell. Biol. 14:59-67).Rat1 IR fibroblasts were cultured in DMEM containing 10% cosmic calfserum and transfected at 80%-90% confluence by the calcium phosphatemethod. Ligands were dissolved in a vehicle containing 0.1 % DMSO (9-cisretinoic acid and troglitazone) or water (8-bromocAMP). Transfectionswere performed in duplicate and repeated at least three times. CATactivity was assayed as described in Kim and Spiegelman ((1996) GenesDev. 10:1096-1107).

For GAL4 fusion constructs, full-length PGC-1 generated by PCR wascloned in-frame into the Sall-EcoRV sites of pCMX-GAL4 plasmid. Murinefull-length SRC-1 was cloned into the Smal site of RSV.GAL4.COS cellswere transfected in the same way as Rat1 IR fibroblasts and the reporterwas the 5xUASg-CAT.

As shown in FIG. 3B, PGC-1 can activate transcription readily whentethered to DNA by the GAL4 DBD. For comparison, the results obtained byfusion of the GAL4 DBD with another coactivator of nuclear receptors,SRC-1, is shown. Thus, PGC-1 does not absolutely require docking to anuclear receptor to demonstrate transcriptional activation function; itis likely that its interaction with these receptors serves primarily tobring PGC-1 to appropriate DNA sites.

To further determine the location of the transcriptional activationdomain of PGC-1, a number of deletion mutants fused to a GAL4 DNAbinding domain were tested for the induction of a luciferase reportergene as described above. The following constructs were tested: controlGAL-4 alone, GAL4-PGC-1, GAL4-amino acids 1-65 of PGC-1, GAL4-aminoacids 1-125 of PGC-1, GAL4-amino acids 1-170 of PGC-1, GAL4-amino acids1-350 of PGC-1, GAL4-amino acids 1-550 of PGC-1, GAL4-amino acids 1-650of PGC-1, GAL4-amino acids 1-650 of PGC-1, and GAL4-amino acids 170-797of PGC-1. The results are summarized below in Table 1.

TABLE 1 Transcription Activity of PGC-1-GAL-4 constructs CONSTRUCTLUCIFERASE UNITS GAL 4 Alone 4 GAL4-PGC-1 700 GAL4 1-65 4800 GAL4 1-12584,000 GAL4 1-170 36,000 GAL4 1-350 700 GAL4 1-550 4,300 GAL4 1-650 300GAL4 170-797 4

As shown in Table 1, GAL4-PGC-1 constructs containing the N-terminalregion of the molecule ) show a higher transcriptional activity than thefull length molecule. The construct GAL4 170-797 showed no detectabletranscriptional activity. These results indicate that thetranscriptional activation domain of PGC-1 is located at the N-terminalregion of the molecule and in particular, at amino acids 1-1 70 ofPGC-1. The decreased in transcriptional activity observed as C-terminalamino acid residues are included (e.g., compare the transcriptionalactivity of GAL4 1-125 and the full length molecule) suggests that theseC-terminal residues may inhibit the transcriptional activity of theN-terminal domain by, e.g., masking this domain or by interacting withother proteins which may mask or otherwise antagonize the activity ofthis domain.

The above-described assay and constructs provide a useful assay forscreening compounds or agents which can modulate, e.g., stimulate orinhibit, the function of PGC-1. Particularly, preferred compounds oragent include activators of PGC-1, e.g., agents that antagonize theinhibitory effect of the C-terminal portion of the molecule. Thesecompounds or agents can be useful in modulating thermogenesis.

Example VIII Role of Protein Kinase a in Modulating PGC-1 Activity

Expression of UCP genes is highly sensitive to cAMP. Analysis of thePGC-1 sequence revealed three consensus sites for phosphorylation byprotein kinase A (FIG. 2A and 2B). This finding suggests a potentialrole of this kinase in regulating the activity of PGC-1, which in tunwould modify UCP gene expression. To address this possibility, sitedirected mutagenesis can be performed to ablate these phosphorylationsites. For example, amino acids 373-376 of SEQ ID NO:2 can be mutatedusing standard protocols. The transcriptional activity of the resultingmutants can be tested in, e.g., COS cells or HeLa cells, carrying areporter gene, e.g., a CAT gene, under the control of an UCP promoter.

Example IX ECTOPIC EXPRESSION OF PGC-1 INDUCES MOLECULAR COMPONENTS OFADAPTIVE THERMOGENESIS

To examine directly the ability of PGC-1 to regulate the genes ofadaptive thermogenesis, retroviral vectors have been used to expressthis protein in white fat precursor cells, and 3T3-F442A preadipocyteswere then stimulated to differentiate. Briefly, the PGC-1 viralexpression vector (pBabe-PGC-1) was constructed by ligating theBamHI-Xhol fragment from pBluescript-PGC-1 plasmid into BamHI/Sall sitesof pBabe-puro. Following drug selection, virally infected 3T3F442A-PGC-1and 3T3F441-vector cells lines were grown to confluence in DMEM with 10%BCS. Differentiation of these cells was initiated by culturing them inDMEM insulin. Cells were refed every 2 days with this medium. Specificcells were grown in DMEM with 10% CCS to confluence. These cells werethen treated with 1 μM dexamethasone, 0.5 mM ofmethyl-isobutyl-xanthine, 125 μM indomethacin, 17 nM insulin, and 1 nMT₃ for 48 hrs. to induce differentiation. Cells were subsequentlymaintained in DMEM containing 10% CCS, 17 nM insulin, and 1 nM T₃ andreplenished every 2 days. After these treatments, total RNA was isolatedand analyzed.

To induce UCP-1 expression, 1 μM 8-bromo-cAMP and 1 mM 9-cis-retinoicacid were added to the medium, and total RNA was extracted from thecells 6 hr later. Northern blot analysis with a PGC-1 probe revealedthat PGC-1 mRNA was barely detectable in these white fat cells infectedwith empty vectors but was more highly expressed in cells infected withthe viruses containing the PGC-1 cDNA. The expression of this mRNA inthe cultured cells was approximately 6% of that seen in the brown fat ofcold exposed mice. mRNA for UCP-1, the classic marker of brown fat cellsthat is encoded in the cell nucleus, is barely detectable in the control3T3-F442A cells but is significantly induced in the cells expressingPGC-1. mRNA for ATP synthetase, a key mitochondrial protein involved inoxidative phosphorylation that is also encoded in the nucleus, islikewise increased in the cells expressing PGC-1. The mitochondrialrespiratory enzyme cytochrome c-oxidase subunits COX II and IV areencoded in the mitochondrial and nuclear genome, respectively. Both ofthese mRNAs increase 2-to 3-fold in the cells ectopically expressingPGC-1. Expression of aP2, a white and brown fat cell gene not linked tothermogenesis, and 36B4, a ribosomal protein are shown as a loadingcontrol. These results demonstrate that PGC-1 can stimulate theexpression of several key genes of mitochondrial function and adaptivethermogenesis, even when expressed at levels far below those seen incold-exposed animals.

The ability of PGC-1 to affect the expression of mRNA for a protein(COX-II) encoded in the mitochondrial genome suggests that PGC-1 couldaffect the biogenesis of mitochondria per se. Changes in the cellularcontent of mitochondrial DNA have been used as a simple biochemicalassay for mitochondrial proliferation (Martin et al. (1995) Biochem. J.308:749-752; Klingenspor et al. (1996) Biochem. J. 316:607-613). Toaddress this possibility, Southern blot analysis of mitochondrial DNAwas performed. 3T3-F442A Southern blots were carried out by isolatingand processing genomic DNA as described in Maniatis et al. (1989)Molecular Cloning: A Laboratory Manual, 2nd Ed. (Cold Spring Harbor,N.Y.: Cold Spring Harbor Laboratory Press)). 3T3-F442A cells weredifferentiated as described above. Total cellular DNA was isolated andwas digested with NCo I. Ten micrograms of DNA were electrophoresed, andthe Southern blot was hybridized using COX-II cDNA as a probe formitochondrial DNA.

Southern blot analysis of mitochondrial genome DNA revealed that cellsexpressing PGC-1 have twice the mitochondrial DNA content compared tocontrol cells. The sane blots were also probed with cDNA for 36B4, aribosomal protein encoded in the nucleus. The blot was then stripped andhybridized with the nuclear gene 3664. These results show that ectopicPGC-1 expression can stimulate an increase in mitochondrial DNA,indicating an increased biogenesis of mitochondria.

Example X Chronic Treatment of PGC-1 Infected Cells Increasing OxygenConsumption

To determine a physiological role for PGC-1 in mediating thermogenesis,oxygen consumption assays were performed using 3T3-F442A preadipocytesinfected with PGC-1-expressing retroviral vectors as described above.The efficiency of infection is estimated to be 25%-30% of the cells.Oxygen consumption assays were performed as described in Ludwik, J. etal. (1981) J. Biochemistry 256(24): 12840-12848 and Hermesh, O. (1998)J. Biochemistry 273(7): 3937-3942. Treatment of these cells with 1 μM8-bromo-cAMP and 1 mM 9-cis-retinoic acid for 6 hours resulted in a 100%increase in oxygen consumption by these cells (FIG. 6). The increase inoxygen consumption detected in these cells is likely to be caused by anincrease in the activity and/or expression of mitochondrial uncouplingproteins (UCPs) or similar proteins which may facilitate protontransport.

These experiments demonstrate that PGC-1 is capable of mediating athermogenic response in vivo, thus linking directly the induction ofmitochondrial DNA and gene expression to a physiological response. Thephysiological function of PGC-1 can be further characterized in tissuesknown to expressed high levels of this molecule, such as muscle. Forexample, mouse myoblast cells which can be induced to differentiate intomyotube such as C2-C 12 cells, can be infected with a retrovirusexpressing PGC-1 and tested under the conditions described above.

Example XI PGC-1 is a Unique Nuclear Receptor Coactivator

The results presented herein show that PGC-1 is unusual among knownnuclear receptor coactivators in that its expression is dramaticallyregulated with respect to both tissue selectivity and the physiologicalstate of the animal. The expression of PGC-1 in BAT but not WATdistinguishes it from most known transcriptional components in thesetissues and its induction by cold is even more dramatic than thatobserved for UCP-1. PGC-1 is also distinct from the known coactivatorsin that it appears to use different sequence motifs for protein-proteindocking, on both sides of the receptor-coactivator pair. Nearly all ofthe known coactivators and corepressors utilize LXXLL (SEQ ID NO:3)sequences to bind at the ligand-regulated helix 12 in the carboxyterminal AF-2 domain (Heery et al. (1997) Nature 387:733-736; Torchia etal. (1997) Nature 387:677-684. In contrast, PGC-1 utilizes a domain richin proline residues to bind to a region that overlaps the DNA bindingand hinge region of PPARγ. For PPARγ, this opens the possibility thatPGC-1 is not an alternative coactivator to one or more of theligand-controlled coactivators but, rather, may bind in concert withthese proteins to give a larger macromolecular complex. On the otherhand, ligand-dependent docking is seen with some other receptors such asthe retinoic acid receptor, the estrogen receptor, and to a certaindegree, the thyroid receptor. Since PGC-1 has one LXXLL (SEQ ID NO:3)sequence, a motif shown in several contexts to be both necessary andsufficient for ligand-dependent receptor docking, it is entirelypossible that the binding of PGC-1 to those receptors will depend onthis sequence and the receptor AF-2 domains.

It is now appreciated that most of the coactivators or corepressors thatbind to receptors at AF-2 domains carry either histone acetyltransferaseor histone deacetylase activities (Pazin and Kadonaga (1997) Cell89:325-328). These activities may be intrinsic to certain coactivatorssuch as CBP and SRC-1 (Bannister and Kouzarides, (1996) Nature384:641-643; Spencer et al. (1997) Nature 389:194-198) or reside inproteins that form complexes with corepressors, as illustrated by thecomplex between SMRT and mammalian histone deacetylase (Nagy et al.(1997) Cell 89:373-380; Torchia et al. (1997) Nature 387:677-684. Basedon primary sequence data, PGC-1 does not contain any motifs that wouldbe suggestive of histone acetylase or deacetylase activity. It also hasno significant sequence homologies with any of the known nuclearreceptor coactivators or corepressors. It may be noteworthy that PGC-1has paired SR and RNA-binding domains that have been identified in anumber of proteins, including several that bind to the regulatorycarboxy terminal domain (CTD) of RNA polymerase II (Yuryev et al. (1996)Proc. Natl. Acad. Sci. USA 93:6975-6980. The findings presented hereincould also be explained by PGC-1 relieving a gene repression mechanism.The hinge region of at least one nuclear receptor (TR) has been shown tobe involved in binding a corepressor (N-CoR; Horlein et al. (1995)Nature 377:397-404. Hence, PGC-1's action may be to derepresstranscription by interfering with corepressor binding.

Example XII role of PGC-1 In Adaptive Thermogenesis

Adaptive thermogenesis refers to a component of energy expenditure,which is separate from physical activity and which can be elevated inresponse to changing environmental conditions, most notably coldexposure and overfeeding (Himms-Hagen (1989) Proc. Soc. Exp. Biol. Med.208:159-169). There is considerable interest in this subject because ofits potential roles in both the pathogenesis and therapy of humanobesity.

A role for PGC-1 in adaptive thermogenesis is indicated first by itsconnection to the key tissues and hormones implicated in this process.The results shown herein suggest an especially important role forskeletal muscle and brown fat. PGC-1 is induced by cold exposure in bothmuscle and brown fat but not in other tissues. The thermogenic andantiobesity properties of brown fat are conclusively established inrodents (Himms-Hagen (1995) Proc. Soc. exp. Biol. Med. 208:159-169), butthe role of BAT is less clear in humans due to the fact that adulthumans and other large mammals do not have well-defined brown fatdepots. The expression of UCP-1 in the white fat depots of adultssuggests that brown adipocytes may be incorporated into depots thatappear white and can be recruited upon adrenergic stimulation (Garrutiand Ricquier, (1992) Int. J. Obes. Relat. Metab. Disord. 16:383-390).

With regard to hormones, thyroid hormone and 0-adrenergic agonistsappear to play the most important roles in both cold and diet-inducedthermogenesis in muscle and brown fat (Himms-Hagen (1989) Proc. Soc.Exp. Biol. Med. 208:259-269; Cannon and Nedergaard (1996) Biochem. Soc.Trans. 24:407-412). β-adrenergic agonists appear to affect PGC-1function in at least two distinct ways. First, they can induce PGC-1expression. Second, cyclic AMP (the intracellular mediator ofβ-adrenergic receptor activity) increases the transcriptional activitymediated by PGC-1 when expression is driven ectopically, as shown inFIG. 3B. While the molecular basis of this is not known, the presence ofthree consensus phosphorylation sites for protein kinase A suggests thatthe protein may be posttranslationally activated by this pathway. Thethermogenic effects of thyroid hormone and its receptors are well known.One of the clearest effects of increasing thyroid hormone levels is thestimulation of mitochondrial respiration rates in skeletal muscle, brownfat, heart, and kidney. Abnormally low respiration rates, characteristicof a hypothyroid state, can be increased by raising thyroid hormonelevels (Pillar and Seitz (1997) Eur. J. Endocrinol. 135:231-239). Basedon the tissues where it is expressed and its ability to coactivate theTR, PGC-1 appears to be a very good candidate to mediate some of theseeffects.

Recent evidence has also suggested interesting effects of the TZDs inthermogenesis. These PPARγ ligands can increase energy expenditure whengiven systematically to rodents, perhaps due to increased formation ofbrown fat and an increase in Ucp-1 gene expression. These effects havealso been seen in cultured cells (Foellmi-Adams et al. (1996) Biochem.Pharmacol. 52:693-701; Tai et al., J. Biol. Chem. 271:29909-29914(1996). The ability of PGC-1 to coactivate the function of PPARγ on theUCP-1 promoter, and presumably other promoters in thermogenic pathways,may provide some explanation for these effects.

In addition to these associations described above, ectopic expressionexperiments presented here show more directly that PGC-1 can regulatecomponents of thermogenesis. At a cellular and molecular level, adaptivethermogenesis consists of at least three separable processes: thebiogenesis of mitochondria, the expression of the mitochondrial enzymesof the respiratory chain, and the expression of specific uncouplingproteins. There are now three known members of the UCP gene family;UCP-1, expressed exclusively in brown fat; Ucp-2, expressed widely, andUcp-3, expressed primarily in skeletal muscle and brown fat. Dependingon the length of time and severity of a given physiological challenge,one or more of these aspects of thermogenesis may be affected in muscle,BAT, or other tissues.

The retroviral expression of PGC-1 described herein have used white fatcells. This cell type was chosen because it has little endogenous PGC-1expression and is known to have relatively low numbers of mitochondriaand little expression of UCP-1 or UCP-3. Although we were only able toget a relatively low level of PGC-1 mRNA expression (6% of that seen incold-induced BAT), it is clear that several molecular components of theadaptive thermogenesis system are altered. First, expression of theUcp-1 gene is turned on from the almost undetectable level that ischaracteristic of these white cells. Second, several mitochondrial genesof the respiratory chain that are ordinarily expressed in these cells,such as ATP synthetase, Cox-II and Cox-IV, are significantly increased.Finally, mitochondrial content is doubled, as evidenced by the increasein mitochondrial DNA per unit of total cellular DNA.

The mechanism by which PGC-1 may regulate mitochondrial processes linkedto adaptive thermogenesis can be as follows. For genes such as UCP-1that are encoded in the nucleus and are responsive to PPAR, TR, or othernuclear receptors, PGC-1 could act directly as a coactivator to increasetranscription rates. For genes that are encoded in the mitochondrialgenome (such as Cox-Il), PGC-1 could be acting directly or indirectly.Certain genes within the mitochondria have been shown to have functionalthyroid response elements (TREs; Pillar and Seitz (1997) Eur. JEndocrinol. 135:231-239). While PGC-1 is observed mainly in the nucleus,a small percentage of the TR and PGC-1 are transported into themitochondria and function directly at these sites. Similarly, withregard to mitochondrial DNA replication, the D loop of the mitochondrialgenome is a site of heavy strand replication and contains a TRE-DR2sequence (Wrutniak et al., (1995) J. Biol. Chem. 270:16347-16354),suggesting that the TR and PGC-1 could act here directly. On the otherhand, PGC-1 and nuclear receptors could regulate the expression of othernuclear factors, such as NRF or mitochondrial factor A, that have beenshown to function in the mitochondria to stimulate gene transcriptionand/or DNA replication (Pillar and Seitz, (1997) Eur. J Endocrinol.135:231-239).

The contents of all cited references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application (including theBackground Section) are hereby expressly incorporated by reference. Theentire contents of Appendix A (including the Figures depicted therein)entitled “A Cold-Inducible Coactivator of Nuclear Receptors Linked toAdaptive Thermogenesis” is also incorporated by reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. An isolated antibody that specifically binds PGC-1.
 2. A method fordetecting the presence of PGC-1 in a biological sample comprisingcontacting a biological sample with an agent capable of detecting PGC-1protein or mRNA.
 3. A kit for detecting the presence of PGC-1 in abiological sample comprising a labeled or labelable agent capable ofdetecting PGC-1 protein or mRNA in a biological sample; means fordetermining the amount of PGC-1 in the sample; and means for comparingthe amount of PGC-1 in the sample with a standard.
 4. A method foridentifying a compound capable of treating a disorder characterized byaberrant PGC-1 nucleic acid expression or PGC-1 protein activitycomprising assaying the ability of the compound or agent to modulate theexpression of PGC-1 nucleic acid or the activity of the PGC-1 proteinthereby identifying a compound capable of treating a disordercharacterized by aberrant PGC-1 nucleic acid expression or PGC-1 proteinactivity.
 5. A method for identifying a compound which binds to PGC-1protein comprising contacting the PGC-1 protein with the compound underconditions which allow binding of the compound to the PGC-1 protein toform a complex; and detecting the formation of a complex of the PGC-1protein and the compound in which the ability of the compound to bind tothe PGC-1 protein is indicated by the presence of the compound in thecomplex.
 6. A method for identifying a compound which inhibits theinteraction of the PGC-1 protein with a target molecule comprisingcontacting, in the presence of the compound, the PGC-1 protein and thetarget molecule under conditions which allow binding of the targetmolecule to the PGC-1 protein to form a complex; and detecting theformation of a complex of the PGC-1 protein and the target molecule inwhich the ability of the compound to inhibit interaction between thePGC-1 protein and the target molecule is indicated by a decrease incomplex formation as compared to the amount of complex formed in theabsence of the compound.